Lipid Formulated Compositions and Methods for Inhibiting Expression of Serum Amyloid A Gene

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) targeting a Serum Amyloid A (SAA) gene, and methods of using the dsRNA to inhibit expression of SAA

FIELD OF THE INVENTION

The invention relates to lipid formulated double-stranded ribonucleicacid (dsRNA) targeting a Serum Amyloid A (SAA) gene, and methods ofusing the dsRNA to inhibit expression of SAA.

BACKGROUND OF THE INVENTION

Serum Amyloid A (SAA) is an 104 amino acid HDL-associated apolipoproteinwhose level in the blood is elevated up to 1000-fold in response tovarious injuries including trauma, inflammation and neoplasia. SAAproteins are involved in cholesterol metabolism and transport,inhibition of lymphocyte and endothelial cell proliferation, inductionof matrix metalloproteinases, and modulation of the inflammatoryresponse via both anti- and pro-inflammatory activities.Pro-inflammatory cytokines, such as IL-1β, IL-6, and TNFα, triggerinflammation and stimulate the production of acute-phase proteins,including SAA1 and SAA2.

Liver is the major site of SAA expression, and extrahepatic SAAexpression has also been described in human atherosclerotic lesions, inthe brains of Alzheimer disease patients, and in synovial tissues fromrheumatoid arthritis patients. SAA levels have also been found to beelevated in the serum of patients with a wide range of malignancies,being highest in those with metastatic carcinoma of unknown primarysites. SAA mRNA and protein has also been found to be locally expressedin human colon carcinoma tissues and in epithelial carcinomas.

Four SAA loci, all mapped to chromosome 11p, have been described. Two ofthe loci (SAA1 and SAA2) encode acute-phase SAAs (A-SAAs), which exhibita dramatic transient increase in serum concentration in response toinflammatory stimuli; a third locus (SAA3) defines a pseudogene; and afourth locus (SAA4) encodes a constitutively expressed SAA (C-SAA),which responds only moderately to inflammatory stimuli. SAA3 isexpressed in mice and other mammalian species, but is not expressed inhumans. SAA1 and SAA2 are 95% homologous in both their coding andnoncoding regions, and are coordinately induced in response toinflammation. The A-SAAs are the circulating precursors of the insolublecleavage product amyloid A that is deposited in major organs insecondary amyloidosis (also called AA amyloidosis, or reactiveamyloidosis), a progressive and fatal disease that is an occasionalconsequence of chronic or episodic inflammatory conditions such asrheumatoid arthritis and leprosy.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.).

SUMMARY OF THE INVENTION

The invention provides compositions containing double-strandedribonucleic acid (dsRNA) and methods for inhibiting the expression of anSAA gene, such as one or both of SAA1 and SAA2, such as in a cell ormammal. The invention also provides compositions and methods fortreating pathological conditions and diseases caused by the expressionof an SAA gene, such as amyloidosis. The dsRNAs included in thecompositions featured herein include a dsRNA having an RNA strand (theantisense strand) having a region that is less than 30 nucleotides inlength, generally 19-24 nucleotides in length, and that is complementaryto at least part of an mRNA transcript of an SAA gene.

In one embodiment, a dsRNA for inhibiting expression of an SAA geneincludes at least two strands that are complementary to each other. ThedsRNA includes a sense strand and an antisense strand. The antisensestrand includes a nucleotide sequence that is complementary to at leastpart of an mRNA encoding SAA, and the region of complementarity is lessthan 30 nucleotides in length, and at least 15 nucleotides in length.Generally, the dsRNA is 19 to 24, e.g., 19 to 21 nucleotides in length.The dsRNA, upon contacting with a cell expressing SAA, inhibits theexpression of an SAA gene by at least 40%, such as when assayed by amethod as described herein.

For example, the dsRNA molecules featured herein can include a sensestrand that is selected from the group consisting of the sense sequencesof Table 2 and an antisense strand that is selected from the groupconsisting of the antisense sequences of Table 2. The dsRNA moleculesfeatured herein can include naturally occurring nucleotides or caninclude at least one modified nucleotide, such as a 2′-O-methyl modifiednucleotide, a nucleotide having a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative. Alternatively,the modified nucleotide may be chosen from the group of: a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide. Generally, such a modifiedsequence will be based on a first sequence of said dsRNA selected fromthe group consisting of the sense sequences of Table 2 and a secondsequence selected from the group consisting of the antisense sequencesof Table 2.

In an embodiment, the dsRNA can include a sense strand including atleast 15 contiguous nucleotides of a sense strand sequence selected fromTable 2. In an embodiment, the dsRNA can include an antisense strandincluding at least 15 contiguous nucleotides of an antisense sequenceselected from Table 2.

In one embodiment, the sense strand can include 15 or more contiguousnucleotides of the nucleotide sequence of SEQ ID NO:37, SEQ ID NO:127,SEQ ID NO:95, SEQ ID NO:105, SEQ ID NO:59, SEQ ID NO:23, SEQ ID NO:155,SEQ ID NO:193, SEQ ID NO:283, SEQ ID NO:251, SEQ ID NO:261, SEQ IDNO:215, SEQ ID NO:179, or SEQ ID NO:311. In an embodiment, the antisensestrand can include 15 or more contiguous nucleotides of the nucleotidesequence of SEQ ID NO:38, SEQ ID NO:128, SEQ ID NO:96, SEQ ID NO:106,SEQ ID NO:60, SEQ ID NO:24, SEQ ID NO:156, SEQ ID NO:194, SEQ ID NO:284,SEQ ID NO:252, SEQ ID NO:262, SEQ ID NO:216, SEQ ID NO:180, or SEQ IDNO:312. In another embodiment, the sense strand can consist of SEQ IDNO:37, SEQ ID NO:127, SEQ ID NO:95, SEQ ID NO:105, SEQ ID NO:59, SEQ IDNO:23, SEQ ID NO:155, SEQ ID NO:193, SEQ ID NO:283, SEQ ID NO:251, SEQID NO:261, SEQ ID NO:215, SEQ ID NO:179, or SEQ ID NO:311 and theantisense strand can consist of SEQ ID NO:38, SEQ ID NO:128, SEQ IDNO:96, SEQ ID NO:106, SEQ ID NO:60, SEQ ID NO:24, SEQ ID NO:156, SEQ IDNO:194, SEQ ID NO:284, SEQ ID NO:252, SEQ ID NO:262, SEQ ID NO:216, SEQID NO:180, or SEQ ID NO:312. In an embodiment, the dsRNA is 18397,18379, 18445, 18420, 18415, 18431, or 18326. In an embodiment, the dsRNAtargets SEQ ID NO:193, SEQ ID NO:283, SEQ ID NO:251, SEQ ID NO:261, SEQID NO:215, SEQ ID NO:179, or SEQ ID NO:311.

In an embodiment, the dsRNA is conjugated to a ligand. In an embodiment,the dsRNA is formulated in a lipid formulation. In an embodiment, thedsRNA is formulated in a LNP formulation, a LNP01 formulation, a LIPIDA-SNALP formulation, or a SNALP formulation.

In an embodiment, administration of the dsRNA to a cell results in about97%, 95%, 92%, 89%, or 74% inhibition of SAA mRNA expression as measuredby a real time PCR assay. In an embodiment, administration of the dsRNAto a cell results in about 89%, 87%, 83%, 68%, or 54% inhibition of SAAmRNA expression as measured by a branched DNA assay. In an embodiment,administration of the dsRNA to a cell results in about 100%, 99%, or 93%inhibition of SAA protein expression as measured by an ELISA assay. Inan embodiment, the dsRNA has an IC50 of less than 10 pM. In anembodiment, administration of the dsRNA reduces SAA protein expressionby about 80% in mice compared to an siRNA control.

In an embodiment, the dsRNA includes an overhang. In an embodiment, thedsRNA includes a dTdT overhang. In an embodiment, the dsRNA comprisestwo dTdT overhangs on the 3′ end of the sense strand and the antisensestrand.

In an embodiment, the sense strand is 21 nucleotides in length. In anembodiment, the antisense strand is 21 nucleotides in length. In anembodiment, the dsRNA comprises one or more2′-O-methylcytidine-5′-phosphates and/or one or more2′-O-methyluridine-5′-phosphates.

In another embodiment, the invention provides a cell containing at leastone of the dsRNAs featured in the invention. The cell is generally amammalian cell, such as a human cell.

In another embodiment, the invention provides a pharmaceuticalcomposition for inhibiting the expression of an SAA gene in an organism,generally a human subject. The composition typically includes one ormore of the dsRNAs described herein and a pharmaceutically acceptablecarrier or delivery vehicle. In one embodiment, the composition is usedfor treating amyloidosis, e.g., AA (secondary or reactive) amyloidosis.

In another embodiment, the pharmaceutical composition is formulated foradministration of a dosage regimen described herein, e.g., not more thanonce every four weeks, not more than once every three weeks, not morethan once every two weeks, or not more than once every week. In anotherembodiment, the pharmaceutical composition can be maintained for a monthor longer, e.g., one, two, three, or six months, or one year or longer.

In another embodiment, a composition containing a dsRNA featured in theinvention, i.e., a dsRNA targeting SAA, is administered with a non-dsRNAtherapeutic agent, such as an agent known to treat amyloidosis, or asymptom of amyloidosis. For example, a dsRNA featured in the inventioncan be administered with an agent for treatment of an inflammatorydisorder, such as chronic inflammatory arthritis, or an agent fortreatment of renal dysfunction. Exemplary agents for treatment ofchronic inflammatory arthritis include anti-cytokine biologics, such asanakinra, tocilizumab, etanercept, infliximab, adlimumab, certolizumab,rituxan, rituximab, chlorambucil, and Eprodisate (Neurochem, Canada).Exemplary agents for treatment of renal dysfunction include, e.g.,diuretics, ACE (Angiotensin-Converting Enzyme) inhibitors, ARBs(angiotensin receptor blocking agents), dialysis in end stage renaldisease (ESRD), and renal transplant.

In another embodiment, an SAA dsRNA is administered to a patient, andthen the non-dsRNA agent is administered to the patient (or vice versa).In another embodiment, an SAA dsRNA and the non-dsRNA therapeutic agentare administered at the same time.

In another embodiment, the invention provides a method for inhibitingthe expression of an SAA gene in a cell by performing the followingsteps:

(a) introducing into the cell a double-stranded ribonucleic acid(dsRNA), wherein the dsRNA includes at least two sequences that arecomplementary to each other. The dsRNA has a sense strand having a firstsequence and an antisense strand having a second sequence; the antisensestrand has a region of complementarity that is complementary to at leasta part of an mRNA encoding SAA, and where the region of complementarityis less than 30 nucleotides in length, generally 19-24 nucleotides inlength, and where the dsRNA, upon contact with a cell expressing an SAA,inhibits expression of an SAA gene by at least 40%;

and

(b) maintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of SAA gene, therebyinhibiting expression of an SAA gene in the cell.

In another embodiment, the method is for inhibiting gene expression in atumor cell.

In another embodiment, the invention provides methods for treating,preventing or managing pathological processes mediated by SAAexpression, such as amyloidosis, e.g., AA amyloidosis. In oneembodiment, the method includes administering to a patient in need ofsuch treatment, prevention or management a therapeutically orprophylactically effective amount of one or more of the dsRNAs featuredin the invention. In one embodiment the patient has amyloidosis. Inanother embodiment, administration of the dsRNA targeting SAA alleviatesor relieves the severity of at least one symptom of an SAA-mediateddisorder in the patient. In one embodiment the patient has psoriaticarthritis, chronic juvenile arthritis, ankylosing spondylitis, Behcet'ssyndrome, Reiter's syndrome, adult Still's disease, inflammatory boweldisease, hereditary periodic fevers, tuberculosis, osteomyelitis,bronchiectasis, leprosy, pyelonephritis, decubitus ulcers, Whipple'sdisease, acne conglobata, common variable immunodeficiencyhypo/agammaglobulinemia, cystic fibrosis, hepatoma, renal carcinoma,Castleman's disease, Hodgkin's disease, adult hairy cell leukemia,Waldenström's disease, a neoplasm, a chronic infections, a chronicinflammatory disease, chronic arthritis, chronic sepsis, a periodicfever syndrome, familial Mediterranean fever, or Crohn's disease.

In another embodiment, the invention provides a vector for inhibitingthe expression of an SAA gene in a cell. In one embodiment, the vectorincludes at least one regulatory sequence operably linked to anucleotide sequence that encodes at least one strand of a dsRNA featuredin the invention.

In another embodiment, the invention provides a cell containing a vectorfor inhibiting the expression of an SAA gene in a cell. The vectorincludes a regulatory sequence operably linked to a nucleotide sequencethat encodes at least one strand of one of the dsRNA featured in theinvention.

In a further embodiment, the invention provides a composition containingan SAA dsRNA, in combination with a second dsRNA targeting a second geneinvolved in a pathological disease, and useful for treating the disease,e.g., amyloidosis.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are graphs showing the effect of IL-β and IL-6 cytokineson SAA mRNA and protein levels in HepB3 cell culture.

FIG. 2 is a bar graph illustrating SAA mRNA levels in Hep3B cellsfollowing administration of candidate SAA siRNAs.

FIG. 3 is a bar graph illustrating SAA protein levels in Hep3B cellsfollowing administration of candidate SAA siRNAs.

FIGS. 4A-4G are graphs illustrating dose response curves for selectedSAA siRNAs.

FIG. 5 is a graph showing that SAA levels were increased in all micetested 24 hours after LPS injection compared to pre-LPS injection SAAlevels.

FIG. 6 is a graph showing that LNP01-formulated 18445 andSNALP-formulated 18445 significantly downregulated SAA levels comparedto controls.

FIG. 7 is a graph showing that expression of hSAA1 can last forapproximately 2 weeks after a single injection of hSAA1-adenovirus.

FIG. 8 is a picture showing a construct for expression of hSAA1 inhepatocytes.

FIG. 9 is a graph showing the expression of hSAA1 in mice followinghydrodynamic injection.

FIG. 10 is a picture showing a construct that was designed for hSAA1transgene expression.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides dsRNAs and methods of using the dsRNAs forinhibiting the expression of an SAA gene in a cell or a mammal where thedsRNA targets an SAA gene. In some embodiments, the dsRNAs featured inthe invention target both an SAA1 gene and an SAA2 gene. The inventionalso provides compositions and methods for treating pathologicalconditions and diseases, such as an amyloidosis, in a mammal caused bythe expression of an SAA gene. dsRNA directs the sequence-specificdegradation of mRNA through a process known as RNA interference (RNAi).

The dsRNAs of the compositions featured herein include an RNA strand(the antisense strand) having a region which is less than 30 nucleotidesin length, generally 19-24 nucleotides in length, and is substantiallycomplementary to at least part of an mRNA transcript of an SAA gene. Theuse of these dsRNAs enables the targeted degradation of mRNAs of genesthat are implicated in pathologies associated with an inflammatoryresponse (e.g., an acute phase inflammatory response) in mammals. Verylow dosages of SAA dsRNAs in particular can specifically and efficientlymediate RNAi, resulting in significant inhibition of expression of anSAA gene. Using cell-based assays, the present inventors havedemonstrated that dsRNAs targeting SAA can specifically and efficientlymediate RNAi, resulting in significant inhibition of expression of anSAA gene. Thus, methods and compositions including these dsRNAs areuseful for treating pathological processes that can be mediated by downregulating SAA, such as in the treatment of amyloidosis.

The dsRNAs of the compositions can include a sense strand including atleast 15, 16, 17, 18, 19, 20, or 21 or more nucleotides of a sensestrand sequence selected from Table 2. The dsRNAs of the compositionscan include an antisense strand including at least 15, 16, 17, 18, 19,20, or 21 or more nucleotides of a sense strand sequence selected fromTable 2. In an embodiment, the sense strand can include 15, 16, 17, 18,19, 20, or 21 or more contiguous nucleotides of SEQ ID NO:37, SEQ IDNO:127, SEQ ID NO:95, SEQ ID NO:105, SEQ ID NO:59, SEQ ID NO:23, or SEQID NO:155. In an embodiment, the antisense strand can include 15, 16,17, 18, 19, 20, or 21 or more contiguous nucleotides of SEQ ID NO:38,SEQ ID NO:128, SEQ ID NO:96, SEQ ID NO:106, SEQ ID NO:60, SEQ ID NO:24,or SEQ ID NO:156.

The dsRNAs of the compositions can target 15, 16, 17, 18, 19, 20, or 21or more contiguous nucleotides of a SAA mRNA, SEQ ID NO:286, SEQ IDNO:220, SEQ ID NO:230, SEQ ID NO:324, SEQ ID NO:223, SEQ ID NO:386,and/or SEQ ID NO:373.

The dsRNA can be conjugated to a ligand. The dsRNA can be formulated ina lipid formulation. In an embodiment, the dsRNA is formulated in a LNPformulation, a LNP01 formulation, a Lipid A-SNALP formulation, or aSNALP formulation.

The dsRNAs of the compositions, when administered to a cell, can resultin about 50-100%, 97%, 95%, 92%, 89%, or 74% inhibition of SAA mRNAexpression as measured by a real time PCR assay. The dsRNAs of thecompositions, when administered to a cell, can result in about 50-100%,89%, 87%, 83%, 68%, or 54% inhibition of SAA mRNA expression as measuredby a branched DNA assay. The dsRNAs of the compositions, whenadministered to a cell, can result in about 50-100%, 100%, 99%, or 93%inhibition of SAA protein expression as measured by an ELISA assay. ThedsRNAs of the compositions have an IC50 of less than 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 pM. The dsRNAs of the compositionscan reduce SAA protein expression by about 40, 50, 60, 70, 80, or 90% inmice compared to an siRNA control.

The methods and compositions containing an SAA dsRNA are useful fortreating pathological processes mediated by SAA expression, such asinflammation-associated disorders, such as amyloidosis. Otherpathological processes can include psoriatic arthritis, chronic juvenilearthritis, ankylosing spondylitis, Behcet's syndrome, Reiter's syndrome,adult Still's disease, inflammatory bowel disease, hereditary periodicfevers, tuberculosis, osteomyelitis, bronchiectasis, leprosy,pyelonephritis, decubitus ulcers, Whipple's disease, acne conglobata,common variable immunodeficiency hypo/agammaglobulinemia, cysticfibrosis, hepatoma, renal carcinoma, Castleman's disease, Hodgkin'sdisease, adult hairy cell leukemia, Waldenström's disease, a neoplasm, achronic infections, a chronic inflammatory disease, chronic arthritis,chronic sepsis, a periodic fever syndrome, familial Mediterranean fever,or Crohn's disease.

The following detailed description discloses how to make and use thecompositions containing dsRNAs to inhibit the expression of an SAA gene,as well as compositions and methods for treating diseases and disorderscaused by the expression of these genes. The pharmaceutical compositionsfeatured in the invention include a dsRNA having an antisense strandcomprising a region of complementarity which is less than 30 nucleotidesin length, generally 19-24 nucleotides in length, and is substantiallycomplementary to at least part of an RNA transcript of an SAA gene,together with a pharmaceutically acceptable carrier. The compositionsfeatured in the invention also include a dsRNA having an antisensestrand having a region of complementarity which is less than 30nucleotides in length, generally 19-24 nucleotides in length, and issubstantially complementary to at least part of an RNA transcript of anSAA gene.

Accordingly, in some aspects, pharmaceutical compositions containing anSAA dsRNA and a pharmaceutically acceptable carrier, methods of usingthe compositions to inhibit expression of an SAA gene, and methods ofusing the pharmaceutical compositions to treat diseases caused byexpression of an SAA gene are featured in the invention.

I. DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dT” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine. However, it will be understood that the term“ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also referto a modified nucleotide, as further detailed below, or a surrogatereplacement moiety. The skilled person is well aware that guanine,cytosine, adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of the invention by a nucleotide containing,for example, inosine. Sequences comprising such replacement moieties areembodiments of the invention.

As used herein, “Serum Amyloid A” (“SAA”) refers to an SAA1 or an SAA2gene (e.g., an endogenous SAA1 or SAA2 gene) in a cell. SAA1 is alsoknown as serum amyloid A1, MGC111216, PIG4, SAA, and tumor protein p53inducible protein 4 (TP53I4). The sequence of two alternative human SAA1mRNA transcripts can be found at NM_(—)000331.3 and NM_(—)199161.2. Thesequence of mouse SAA1 mRNA can be found at NM_(—)009117.3. A single,near full length, SAA-like trace cDNA sequence from cynomolgus monkey isMfa#S27795076 (Macaca fascicularis).

SAA2 is also known as serum amyloid A2 and SAA. The sequence of twoalternative human SAA2 mRNA transcripts can be found atNM_(—)001127380.1 and NM_(—)030754.3. The sequence of mouse SAA2 mRNA isat NM_(—)011314.1.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an SAA gene, including mRNA that is a product of RNA processing of aprimary transcription product. The target sequence is complementary tothe dsRNA antisense sequence and thus has the same sequence as the dsRNAsense sequence, minus any overhang that is present in the sense strand.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding SAA, such as SAA1 or SAA2) including a5′ UTR, an open reading frame (ORF), or a 3′ UTR. For example, apolynucleotide is complementary to at least a part of an SAA mRNA if thesequence is substantially complementary to a non-interrupted portion ofan mRNA encoding SAA.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. In general, the majority of nucleotides ofeach strand are ribonucleotides, but as described in detail herein, eachor both strands can also include at least one non-ribonucleotide, e.g.,a deoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, “dsRNA” may include chemical modifications toribonucleotides, including substantial modifications at multiplenucleotides and including all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an siRNA typemolecule, are encompassed by “dsRNA” for the purposes of thisspecification and claims.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′ end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” Where the two strands are connected covalently by means otherthan an uninterrupted chain of nucleotides between the 3′-end of onestrand and the 5′ end of the respective other strand forming the duplexstructure, the connecting structure is referred to as a “linker.” TheRNA strands may have the same or a different number of nucleotides. Themaximum number of base pairs is the number of nucleotides in theshortest strand of the dsRNA minus any overhangs that are present in theduplex. In addition to the duplex structure, a dsRNA may comprise one ormore nucleotide overhangs. The term “siRNA” is also used herein to referto a dsRNA as described above.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule. In an embodiment, the sequences shown in the“Sequence without chemistry (5′-3′)” column of Table 2 (SAA siRNAs;below) can include one or more overhangs comprised of one or morenucleotides. In one aspect, the overhang is a two nucleotide 3′ overhangcomprising the sequence NN, where NN can be any nucleotide, e.g., C, A,G, T. In an embodiment, the overhang can include one or morephosphorothioates on the overhang, e.g., the terminal 3′ dT of theoverhang can have a phosphorothioate. In an embodiment, the overhang isdTsdT.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

“Introducing into a cell,” when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell,” wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of” and the like in as far asthey refer to an SAA gene, herein refer to the at least partialsuppression of the expression of an SAA gene, as manifested by areduction of the amount of mRNA which may be isolated and/or detectedfrom a first cell or group of cells in which an SAA gene is transcribedand which has or have been treated such that the expression of an SAAgene is inhibited, as compared to a second cell or group of cellssubstantially identical to the first cell or group of cells but whichhas or have not been so treated (control cells). The degree ofinhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to SAA genetranscription, e.g., the amount of protein encoded by an SAA gene whichis secreted by a cell, or the number of cells displaying a certainphenotype, e.g., apoptosis. In principle, SAA gene silencing may bedetermined in any cell expressing the target, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given dsRNA inhibitsthe expression of an SAA gene by a certain degree and therefore isencompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

For example, in certain instances, expression of an SAA gene issuppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% by administration of the double-stranded oligonucleotide featuredin the invention. In some embodiments, an SAA gene is suppressed by atleast about 60%, 70%, or 80% by administration of the double-strandedoligonucleotide featured in the invention. In some embodiments, an SAAgene is suppressed by at least about 85%, 90%, or 95% by administrationof the double-stranded oligonucleotide featured in the invention. Tables3, 4, and 5, and FIGS. 2 and 3 indicate a range of inhibition ofexpression obtained in in vitro and ex vivo assays using various SAAdsRNA molecules at various concentrations.

As used herein in the context of SAA expression, the terms “treat,”“treatment,” and the like, refer to relief from or alleviation ofpathological processes mediated by SAA expression. In the context of thepresent invention insofar as it relates to any of the other conditionsrecited herein below (other than pathological processes mediated by SAAexpression), the terms “treat,” “treatment,” and the like mean torelieve or alleviate at least one symptom associated with suchcondition, or to slow or reverse the progression of such condition, suchas the slowing and progression of amyloidosis.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes mediated by SAA expression or an overt symptom ofpathological processes mediated by SAA expression. The specific amountthat is therapeutically effective can be readily determined by anordinary medical practitioner, and may vary depending on factors knownin the art, such as, for example, the type of pathological processesmediated by SAA expression, the patient's history and age, the stage ofpathological processes mediated by SAA expression, and theadministration of other anti-pathological processes mediated by SAAexpression agents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of a RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter. For example, a therapeuticallyeffective amount of a dsRNA targeting SAA can reduce SAA serum levels byat least 25%. In another example, a therapeutically effective amount ofa dsRNA targeting SAA can improve renal function by at least 25%.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. DOUBLE-STRANDED RIBONUCLEIC ACID (DSRNA)

As described in more detail herein, the invention providesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of an SAA gene in a cell or mammal, e.g., in a human havingan amyloidosis, where the dsRNA includes an antisense strand having aregion of complementarity which is complementary to at least a part ofan mRNA formed in the expression of an SAA gene, and where the region ofcomplementarity is less than 30 nucleotides in length, generally 19-24nucleotides in length, and where said dsRNA, upon contact with a cellexpressing said SAA gene, inhibits the expression of said SAA gene by atleast 30% as assayed by, for example, a PCR or branched DNA (bDNA)-basedmethod, or by a protein-based method, such as by Western blot.Expression of an SAA gene can be reduced by at least 30% when measuredby an assay as described in the Examples below. For example, expressionof an SAA gene in cell culture, such as in HepB3 cells, can be assayedby measuring SAA mRNA levels, such as by bDNA or TaqMan assay, or bymeasuring protein levels, such as by ELISA assay. The dsRNA of theinvention can further include one or more single-stranded nucleotideoverhangs.

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc. The dsRNA includes two RNA strands that aresufficiently complementary to hybridize to form a duplex structure. Onestrand of the dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence, derived from the sequence of anmRNA formed during the expression of an SAA gene, the other strand (thesense strand) includes a region that is complementary to the antisensestrand, such that the two strands hybridize and form a duplex structurewhen combined under suitable conditions. Optionally, the region of theantisense strand that is substantially complementary to a sequence of anSAA mRNA is substantially complementary to both an SAA1 and an SAA2mRNA. Generally, the duplex structure is between 15 and 30, moregenerally between 18 and 25, yet more generally between 19 and 24, andmost generally between 19 and 21 base pairs in length. Similarly, theregion of complementarity to the target sequence is between 15 and 30,or between 25 and 30, or between 18 and 25, or between 19 and 24, orbetween 19 and 21, or 19, 20, or 21 base pairs in length. In oneembodiment the duplex is 19 base pairs in length. In another embodimentthe duplex is 21 base pairs in length. When two different siRNAs areused in combination, the duplex lengths can be identical or can differ.

Each strand of the dsRNA of invention is generally between 15 and 30, orbetween 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides inlength. In other embodiments, each is strand is 25-30 nucleotides inlength. Each strand of the duplex can be the same length or of differentlengths. When two different siRNAs are used in combination, the lengthsof each strand of each siRNA can be identical or can differ.

The dsRNA of the invention can include one or more single-strandedoverhang(s) of one or more nucleotides. In one embodiment, at least oneend of the dsRNA has a single-stranded nucleotide overhang of 1 to 4,generally 1 or 2 nucleotides. In another embodiment, the antisensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the sense strand. In further embodiments, the sensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the antisense strand.

‘Generally, the dsRNA includes two 3’ overhangs. In an embodiment, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. In another embodiment, the sense strand of thedsRNA has a nucleotide overhang at the 3′ end and the 5′ end is blunt.In another embodiment, both ends of the dsRNA can be blunt. In anotherembodiment, one or more of the nucleotides in the overhang is replacedwith a nucleoside thiophosphate.

In one embodiment, an SAA gene is a human SAA gene. In specificembodiments, the sense strand of the dsRNA is one of the sense sequencesfrom Table 2, and the antisense strand is one of the antisense sequencesof Table 2. Alternative antisense agents that target elsewhere in thetarget sequence provided in Table 2 can readily be determined using thetarget sequence and the flanking SAA sequence.

The skilled person is well aware that dsRNAs having a duplex structureof between 20 and 23, but specifically 21, base pairs have been hailedas particularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger dsRNAs can be effective as well. In the embodiments describedabove, by virtue of the nature of the oligonucleotide sequences providedin Table 2, the dsRNAs featured in the invention can include at leastone strand of a length described therein. It can be reasonably expectedthat shorter dsRNAs having one of the sequences of Table 2 minus only afew nucleotides on one or both ends may be similarly effective ascompared to the dsRNAs described above. Hence, dsRNAs having a partialsequence of at least 15, 16, 17, 18, 19, 20, 21, or 22 or morecontiguous nucleotides from one of the sequences of Table 2, anddiffering in their ability to inhibit the expression of an SAA gene inan assay as described herein below by not more than 5, 10, 15, 20, 25,or 30% inhibition from a dsRNA comprising the full sequence, arecontemplated by the invention. Further, dsRNAs that cleave within adesired SAA target sequence can readily be made using the correspondingSAA antisense sequence and a complementary sense sequence.

In addition, the dsRNAs provided in Table 2 identify a site in an SAAmRNA (e.g., in an SAA1 and/or an SAA2 mRNA) that is susceptible to RNAibased cleavage. As such, the present invention further features dsRNAsthat target within the sequence targeted by one of the agents of thepresent invention. As used herein, a second dsRNA is said to targetwithin the sequence of a first dsRNA if the second dsRNA cleaves themessage anywhere within the mRNA that is complementary to the antisensestrand of the first dsRNA. Such a second dsRNA will generally consist ofat least 15 contiguous nucleotides from one of the sequences provided inTable 2 coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in an SAA1 or SAA2 gene. Forexample, the last 15 nucleotides of SEQ ID NO:1 combined with the nextsix nucleotides from the target SAA gene produces a single strand agentof 21 nucleotides that is based on one of the sequences provided inTable 2.

The dsRNA featured in the invention can contain one or more mismatchesto the target sequence. In one embodiment, the dsRNA featured theinvention contains no more than 3 mismatches. If the antisense strand ofthe dsRNA contains mismatches to a target sequence, it is preferablethat the area of mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of an SAA gene, the dsRNA generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed within the invention can be used to determine whether a dsRNAcontaining a mismatch to a target sequence is effective in inhibitingthe expression of an SAA gene. Consideration of the efficacy of dsRNAswith mismatches in inhibiting expression of an SAA gene is important,especially if the particular region of complementarity in an SAA gene isknown to have polymorphic sequence variation within the population.

Modifications

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids featured in the invention may besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Specific examplesof dsRNA compounds useful in this invention include dsRNAs containingmodified backbones or no natural internucleoside linkages. As defined inthis specification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Modified dsRNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Modified dsRNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or ore or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other suitable dsRNA mimetics, both the sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,a dsRNA mimetic that has been shown to have excellent hybridizationproperties, is referred to as a peptide nucleic acid (PNA). In PNAcompounds, the sugar backbone of a dsRNA is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representative U.S.patents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Further teaching of PNAcompounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Other embodiments of the invention are dsRNAs with phosphorothioatebackbones and oligonucleosides with heteroatom backbones, and inparticular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene(methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Preferred dsRNAs comprise one of the following at the 2′ position: OH;F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred dsRNAs comprise one of the following at the 2′ position:C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3,SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an dsRNA, or a group for improving thepharmacodynamic properties of an dsRNA, and other substituents havingsimilar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxygroup. A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH2)2, also described in examples herein below.

Other preferred modifications include 2′-methoxy (2′-OCH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on the dsRNA,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.DsRNAs may also have sugar mimetics such as cyclobutyl moieties in placeof the pentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

dsRNAs may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Conjugates

Another modification of the dsRNAs featured in the invention involveschemically linking to the dsRNA one or more moieties or conjugates whichenhance the activity, cellular distribution or cellular uptake of thedsRNA. Such moieties include but are not limited to lipid moieties suchas a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA,1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem.Let., 1994, 4:1053-1060), a thioether, e.g., beryl-5-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan etal., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990,259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

Representative U.S. patents that teach the preparation of such dsRNAconjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within a dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a dsRNA compound. These dsRNAs typically contain at leastone region wherein the dsRNA is modified so as to confer upon the dsRNAincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RnaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of Rnase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of dsRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter dsRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxydsRNAs hybridizing to the same target region.Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such dsRNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of dsRNAs bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the dsRNA still bound to the solid support orfollowing cleavage of the dsRNA in solution phase. Purification of thedsRNA conjugate by HPLC typically affords the pure conjugate.

Vector Encoded dsRNAs

In another aspect, SAA dsRNA molecules are expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be incorporated and inherited as a transgeneintegrated into the host genome. The transgene can also be constructedto permit it to be inherited as an extrachromosomal plasmid (Gassmann,et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. Inone embodiment, a dsRNA is expressed as an inverted repeat joined by alinker polynucleotide sequence such that the dsRNA has a stem and loopstructure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. Et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors featured in the invention can bepseudotyped with surface proteins from vesicular stomatitis virus (VSV),rabies, Ebola, Mokola, and the like. AAV vectors featured in theinvention can be made to target different cells by engineering thevectors to express different capsid protein serotypes. For example, anAAV vector expressing a serotype 2 capsid on a serotype 2 genome iscalled AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can bereplaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.Techniques for constructing AAV vectors which express different capsidprotein serotypes are within the skill in the art; see, e.g., RabinowitzJ E et al. (2002), J Virol 76:791-801, the entire disclosure of which isherein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Viral vectors can be derived from AV and AAV. In one embodiment, thedsRNA featured in the invention is expressed as two separate,complementary single-stranded RNA molecules from a recombinant AAVvector having, for example, either the U6 or H1 RNA promoters, or thecytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA featured in the invention,a method for constructing the recombinant AV vector, and a method fordelivering the vector into target cells, are described in Xia H et al.(2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA featured in the invention,methods for constructing the recombinant AV vector, and methods fordelivering the vectors into target cells are described in Samulski R etal. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol,70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are herein incorporated byreference.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector featured in the invention may be a eukaryotic RNA polymerase I(e.g., ribosomal RNA promoter), RNA polymerase II (e.g., CMV earlypromoter or actin promoter or U1 snRNA promoter) or generally RNApolymerase III promoter (e.g., U6 snRNA or 7SK RNA promoter) or aprokaryotic promoter, for example the T7 promoter, provided theexpression plasmid also encodes T7 RNA polymerase required fortranscription from a T7 promoter. The promoter can also direct transgeneexpression to the pancreas (see, e.g., the insulin regulatory sequencefor pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g., Oligofectamine)or non-cationic lipid-based carriers (e.g., Transit-TKOTM). Multiplelipid transfections for dsRNA-mediated knockdowns targeting differentregions of a single SAA gene or multiple SAA genes over a period of aweek or more are also contemplated by the invention. Successfulintroduction of vectors into host cells can be monitored using variousknown methods. For example, transient transfection can be signaled witha reporter, such as a fluorescent marker, such as Green FluorescentProtein (GFP). Stable transfection of cells ex vivo can be ensured usingmarkers that provide the transfected cell with resistance to specificenvironmental factors (e.g., antibiotics and drugs), such as hygromycinB resistance.

SAA specific dsRNA molecules can also be inserted into vectors and usedas gene therapy vectors for human patients. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or caninclude a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

III. PHARMACEUTICAL COMPOSITIONS CONTAINING DSRNA

In one embodiment, the invention provides pharmaceutical compositionscontaining a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition containing the dsRNAis useful for treating a disease or disorder associated with theexpression or activity of an SAA gene, such as pathological processesmediated by SAA expression. Such pharmaceutical compositions areformulated based on the mode of delivery. One example is compositionsthat are formulated for systemic administration via parenteral delivery,e.g., by intravenous (IV) delivery. Another example is compositions thatare formulated for direct delivery into the brain parenchyma, e.g., byinfusion into the brain, such as by continuous pump infusion.

In general, a suitable dose of dsRNA will be in the range of 0.01 to200.0 milligrams per kilogram body weight of the recipient per day,generally in the range of 0.1 to 50 or 0.1 to 5.0 mg per kilogram bodyweight per day. For example, the dsRNA can be administered at 0.01mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg,1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8mg/kg, 1.9 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30mg/kg, 40 mg/kg, or 50 mg/kg per single dose. The pharmaceuticalcomposition may be administered once daily or the dsRNA may beadministered as two, three, or more sub-doses at appropriate intervalsthroughout the day or even using continuous infusion or delivery througha controlled release formulation. In that case, the dsRNA contained ineach sub-dose must be correspondingly smaller in order to achieve thetotal daily dosage. The dosage unit can also be compounded for deliveryover several days, e.g., using a conventional sustained releaseformulation which provides sustained release of the dsRNA over a severalday period. Sustained release formulations are well known in the art andare particularly useful for delivery of agents at a particular site,such as could be used with the agents of the present invention. In thisembodiment, the dosage unit contains a corresponding multiple of thedaily dose.

The effect of a single dose on SAA levels (or both SAA1 and SAA2 levels)is long lasting, such that subsequent doses are administered at not morethan 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 weekintervals.

The present invention includes pharmaceutical compositions that can bedelivered by injection directly into the brain. The injection can be bystereotactic injection into a particular region of the brain (e.g., thesubstantia nigra, cortex, hippocampus, striatum, or globus pallidus), orthe dsRNA can be delivered into multiple regions of the central nervoussystem (e.g., into multiple regions of the brain, and/or into the spinalcord). The dsRNA can also be delivered into diffuse regions of the brain(e.g., diffuse delivery to the cortex of the brain).

In one embodiment, a dsRNA targeting SAA can be delivered by way of acannula or other delivery device having one end implanted in a tissue,e.g., the brain, e.g., the substantia nigra, cortex, hippocampus,striatum, corpus callosum or globus pallidus of the brain. The cannulacan be connected to a reservoir of the dsRNA composition. The flow ordelivery can be mediated by a pump, e.g., an osmotic pump or minipump,such as an Alzet pump (Durect, Cupertino, Calif.). In one embodiment, apump and reservoir are implanted in an area distant from the tissue,e.g., in the abdomen, and delivery is effected by a conduit leading fromthe pump or reservoir to the site of release. Infusion of the dsRNAcomposition into the brain can be over several hours or for severaldays, e.g., for 1, 2, 3, 5, or 7 days or more. Devices for delivery tothe brain are described, for example, in U.S. Pat. Nos. 6,093,180, and5,814,014.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by SAA expression. Such models are used for in vivo testing ofdsRNA, as well as for determining a therapeutically effective dose. Asuitable mouse model is, for example, a mouse containing a plasmidexpressing human SAA1 or SAA2, e.g., from an adenoviral vector. Anothersuitable mouse model is a transgenic mouse carrying a transgene thatexpresses human SAA1 or SAA2.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

The dsRNAs featured in the invention can be administered in combinationwith other known agents effective in treatment of pathological processesmediated by target gene expression. In any event, the administeringphysician can adjust the amount and timing of dsRNA administration onthe basis of results observed using standard measures of efficacy knownin the art or described herein.

Administration

The present invention also includes pharmaceutical compositions andformulations which include the dsRNA compounds featured in theinvention. The pharmaceutical compositions of the present invention maybe administered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical, pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intraparenchymal, intrathecal orintraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Suitable topical formulations include those inwhich the dsRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively, dsRNAs maybe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC1-10 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancers,surfactants, and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention may be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Liposomal Formulations

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and Novasome™II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether)were used to deliver cyclosporin-A into the dermis of mouse skin.Results indicated that such non-ionic liposomal systems were effectivein facilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside GM1, galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside GM1 or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C1215G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

SNALPs

In one embodiment, a dsRNA featured in the invention is fullyencapsulated in the lipid formulation to form a SPLP, pSPLP, SNALP, orother nucleic acid-lipid particle. As used herein, the term “SNALP”refers to a stable nucleic acid-lipid particle, including SPLP. As usedherein, the term “SPLP” refers to a nucleic acid-lipid particlecomprising plasmid DNA encapsulated within a lipid vesicle. SNALPs andSPLPs typically contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). SNALPs and SPLPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). SPLPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The particlesof the present invention typically have a mean diameter of about 50 nmto about 150 nm, more typically about 60 nm to about 130 nm, moretypically about 70 nm to about 110 nm, most typically about 70 to about90 nm, and are substantially nontoxic. In addition, the nucleic acidswhen present in the nucleic acid-lipid particles of the presentinvention are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1, or 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 11:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (Dlin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (Dlin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (Dlin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (Dlin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (Dlin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (Dlin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (Dlin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (Dlin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DlinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (Dlin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (Dlin-K-DMA) oranalogs thereof, or a mixture thereof. The cationic lipid may comprisefrom about 20 mol % to about 60 mol % or about 40 mol %, 50 mol %, 51mol %, 52 mol %, 53 mol %, 54 mol %, 55 mol %, 56 mol %, 57 mol %, 58mol %, 59 mol %, or 60 mol %, of the total lipid present in theparticle.

In another embodiment, the cationic lipid2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Lipid A) can beused to prepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Lipid A) isdescribed in U.S. provisional patent application No. 61/107,998 filed onOct. 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40%2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Lipid A): 10% DSPC:40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid may be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle. In someembodiments the non-ationic lipid is around from about 7 mol % to about8 mol %, or 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 mol%.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles may be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

LNP01

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (Formula 1),Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids)can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01particles). Stock solutions of each in ethanol can be prepared asfollows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions canthen be combined in a, e.g., 42:48:10 molar ratio. The combined lipidsolution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5)such that the final ethanol concentration is about 35-45% and the finalsodium acetate concentration is about 100-300 mM. Lipid-siRNAnanoparticles typically form spontaneously upon mixing. Depending on thedesired particle size distribution, the resultant nanoparticle mixturecan be extruded through a polycarbonate membrane (e.g., 100 nm cut-off)using, for example, a thermobarrel extruder, such as Lipex Extruder(Northern Lipids, Inc). In some cases, the extrusion step can beomitted. Ethanol removal and simultaneous buffer exchange can beaccomplished by, for example, dialysis or tangential flow filtration.Buffer can be exchanged with, for example, phosphate buffered saline(PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1,about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-siRNA formulations are as follows:

cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugateCationic Lipid Lipid:siRNA ratio Process SNALP 1,2-Dilinolenyloxy-DLinDMA/DPPC/Cholesterol/PEG- N,N- cDMA dimethylaminopropane(57.1/7.1/34.4/1.4) (DLinDMA) lipid:siRNA ~7:1 SNALP- 2,2-Dilinoleyl-4-LIPID A/DPPC/Cholesterol/PEG- LIPID A dimethylaminoethyl- cDMA[1,3]-dioxolane 57.1/7.1/34.4/1.4 (LIPID A) lipid:siRNA ~7:1 LNP052,2-Dilinoleyl-4- LIPID A/DSPC/Cholesterol/PEG- Extrusiondimethylaminoethyl- DMG [1,3]-dioxolane 57.5/7.5/31.5/3.5 (LIPID A)lipid:siRNA ~6:1 LNP06 2,2-Dilinoleyl-4- LIPID A/DSPC/Cholesterol/PEG-Extrusion dimethylaminoethyl- DMG [1,3]-dioxolane 57.5/7.5/31.5/3.5(LIPID A) lipid:siRNA ~11:1 LNP07 2,2-Dilinoleyl-4- LIPIDA/DSPC/Cholesterol/PEG- In-line dimethylaminoethyl- DMG mixing[1,3]-dioxolane 60/7.5/31/1.5, (LIPID A) lipid:siRNA ~6:1 LNP082,2-Dilinoleyl-4- LIPID A/DSPC/Cholesterol/PEG- In-linedimethylaminoethyl- DMG mixing [1,3]-dioxolane 60/7.5/31/1.5, (LIPID A)lipid:siRNA ~11:1 LNP09 2,2-Dilinoleyl-4- LIPID A/DSPC/Cholesterol/PEG-In-line dimethylaminoethyl- DMG mixing [1,3]-dioxolane 50/10/38.5/1.5(LIPID A) Lipid:siRNA 10:1 LNP10 (3aR,5s,6a5)-N,N-ALN100/DSPC/Cholesterol/PEG- In-line dimethyl-2,2- DMG mixingdi((9Z,12Z)- 50/10/38.5/1.5 octadeca-9,12- Lipid:siRNA 10:1dienyl)tetrahydro- 3aH- cyclopenta[d][1,3]dioxol- 5-amine (ALN100) LNP11(6Z,9Z,28Z,31Z)- MC-3/DSPC/Cholesterol/PEG-DMG In-line heptatriaconta-50/10/38.5/1.5 mixing 6,9,28,31-tetraen- Lipid:siRNA 10:1 19-yl 4-(dimethylamino)butanoate (MC3) LNP12 1,1′-(2-(4-(2-((2- TechG1/DSPC/Cholesterol/PEG- In-line (bis(2- DMG mixinghydroxydodecyl)amino)ethyl) (2- 50/10/38.5/1.5hydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:1 1-yl)ethylazanediyl)didodecan- 2-ol (Tech G1)

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising two immiscible liquid phases intimately mixed and dispersedwith each other. In general, emulsions may be of either the water-in-oil(w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finelydivided into and dispersed as minute droplets into a bulk oily phase,the resulting composition is called a water-in-oil (w/o) emulsion.Alternatively, when an oily phase is finely divided into and dispersedas minute droplets into a bulk aqueous phase, the resulting compositionis called an oil-in-water (o/w) emulsion. Emulsions may containadditional components in addition to the dispersed phases, and theactive drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of ease of formulation, as well as efficacyfrom an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions of dsRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the dsRNAs and nucleicacids of the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to affect the efficient delivery of nucleic acids,particularly dsRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of dsRNAs through the mucosa isenhanced. In addition to bile salts and fatty acids, these penetrationenhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyland t-butyl), and mono- and di-glycerides thereof (i.e., oleate,laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44,651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9^(th) Ed., Hardman et al. Eds., McGraw-Hill, New York,1996, pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. Suitable bile salts include, forexample, cholic acid (or its pharmaceutically acceptable sodium salt,sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholicacid (sodium deoxycholate), glucholic acid (sodium glucholate),glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of dsRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as Dnase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Suitable chelating agents include butare not limited to disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines)(Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33;Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of dsRNAs throughthe alimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more dsRNA compounds and (b) one or moreanti-cytokine biologic agents which function by a non-RNAi mechanism.Examples of such biologics include, biologics that target IL1β (e.g.,anakinra), IL6 (tocilizumab), or TNF (etanercept, infliximab, adlimumab,or certolizumab).

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more dsRNA compounds and (b) one or moreother chemotherapeutic agents which function by a non-RNAi mechanism.Examples of such chemotherapeutic agents include but are not limited todaunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphor-amide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FudR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy,15^(th) Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. Whenused with the dsRNAs featured in the invention, such chemotherapeuticagents may be used individually (e.g., 5-FU and oligonucleotide),sequentially (e.g., 5-FU and oligonucleotide for a period of timefollowed by MTX and oligonucleotide), or in combination with one or moreother such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide,or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs,including but not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions featured in the invention. See, generally, The MerckManual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987,Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-RNAichemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulation a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the dsRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby SAA expression. In any event, the administering physician can adjustthe amount and timing of dsRNA administration on the basis of resultsobserved using standard measures of efficacy known in the art ordescribed herein.

Methods for Treating Diseases Caused by Expression of an SAA Gene

The invention relates in particular to the use of a dsRNA targeting SAAand compositions containing at least one such dsRNA for the treatment ofan SAA-mediated disorder or disease. For example, a dsRNA targeting anSAA gene, e.g., one or both of SAA1 and SAA2, can be useful for thetreatment of a disorder associated with inflammation, such as arthritis(e.g., rheumatoid arthritis), or tissue injury, reactive (secondary)amyloidosis or systemic amyloidosis, atherosclerosis, or Alzheimer'sDisease.

A dsRNA targeting an SAA gene is also used for treatment of symptoms anddisorders, such as chronic inflammatory diseases, chronic infections,and neoplasia. Such disorders are frequently associated withamyloidosis. Examples of chronic inflammatory diseases includerheumatoid arthritis, psoriatic arthritis, chronic juvenile arthritis,ankylosing spondylitis, Behcet's syndrome, Reiter's syndrome, AdultStill's disease, inflammatory bowel disease (e.g., Crohn's disease), andhereditary periodic fevers, such as Familial Mediterranean fever.Examples of chronic infections associated with amyloidosis, and suitablefor treatment with SAA dsRNAs, include tuberculosis, osteomyelitis,bronchiectasis, leprosy, pyelonephritis, decubitus ulcers, Whipple'sdisease, acne conglobata, common variable immunodeficiencyhypo/agammaglobulinemia, cystic fibrosis. Examples of neoplasiaassociated with amyloidosis, and suitable for treatment with SAA dsRNAs,include hepatoma, renal carcinoma, Castleman's disease, Hodgkin'sdisease, Adult hairy cell leukemia, and Waldenström's disease.

In one embodiment, a dsRNA targeting an SAA gene is used to treatclinical disorders such as proteinuria/renal insufficiency,diarrhea/obstipation/malabsorption, goiter, neuropathy/carpal tunnelsyndrome, hepatomegaly, lymphadenopathy, cardiac. These disorders arefrequently present in patients with amyloidosis.

A dsRNA targeting an SAA gene can also be used to treat a proliferativedisorder, such as cancer, such as colon cancer. A composition containinga dsRNA targeting an SAA gene is also used to treat a carcinoma of thebreast, ovary, cervix, kidney, or a squamous cell.

A composition containing a dsRNA targeting SAA, e.g., one or both ofSAA1 or SAA2, may also be used to treat other tumors and cancers, suchas breast cancer, lung cancer, head and neck cancer, brain cancer,abdominal cancer, colon cancer, colorectal cancer, esophagus cancer,gastrointestinal cancer, tongue cancer, neuroblastoma, osteosarcoma,ovarian cancer, pancreatic cancer, prostate cancer, cervical cancer(e.g., squamous carcinoma of the cervix), lymphoid tumor,retinoblastoma, Wilm's tumor, multiple myeloma and for the treatment ofskin cancer, like melanoma, for the treatment of lymphomas and bloodcancer. The compositions featured herein can be used to treat a tumor ofthe brain or spine.

A dsRNA targeting SAA may be used to treat a proliferative disorder ordifferentiative disorder. Examples of cellular proliferative and/ordifferentiative disorders include cancer, e.g., carcinoma, sarcoma,metastatic disorders or hematopoietic neoplastic disorders, e.g.,leukemias. A metastatic tumor can arise from a multitude of primarytumor types, including those of prostate, colon, lung, breast and liverorigin. As used herein, the terms “cancer,” “hyperproliferative,” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state of condition characterized by rapidlyproliferating cell growth. These terms are meant to include all types ofcancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. Proliferative disordersalso include hematopoietic neoplastic disorders, including diseasesinvolving hyperplastic/neoplastic cells of hematopoictic origin, e.g.,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof.

Owing to the inhibitory effects on SAA expression, a compositionaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life.

The invention further relates to the use of a dsRNA or a pharmaceuticalcomposition thereof, e.g., for treating an amyloidosis, in combinationwith other pharmaceuticals and/or other therapeutic methods, e.g., withknown pharmaceuticals and/or known therapeutic methods, such as, forexample, those which are currently employed for treating this disorders.In one example, a dsRNA targeting SAA can be administered in combinationwith an anti-cytokine agent such as an anti-IL1β agent (e.g., anakinra),IL6 agent (e.g., tocilizumab), or TNFα agent (e.g., etanercept,infliximab, adlimumab, or certolizumab). In other examples, a dsRNAtargeting SAA can be administered in combination with rituxan(rituximab), Eprodisate (Neurochem, Canada). In yet other examples, adsRNA targeting SAA can be administered in combination with steroids ormethotrexate, e.g., to manage chronic inflammatory arthritis. In otherexamples, a dsRNA targeting SAA can be administered in combination withdiuretics, ACE inhibitors, or ARBs, e.g., for management of renalfunction.

The invention further relates to the use of a dsRNA or a pharmaceuticalcomposition thereof, e.g., for treating a cancer, in combination withother pharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating these disorders. In oneexample, administration of a dsRNA targeting SAA can be administered incombination with a chemotherapeutic agent, such as temozolomide,deoxycoformycin, cisplatin, cyclophosphamide, 5-fluorouracil,adriamycin, daunorubicin, tamoxifen aunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FudR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy,15^(th) Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. Whenused with the dsRNAs featured in the invention, such chemotherapeuticagents may be used individually, sequentially (e.g., dsRNA for a periodof time, followed by chemotherapy), or in combination with one or moreother such agents (e.g., chemotherapy and dsRNA). Two or more combinedcompounds may be used together or sequentially.

The dsRNA and an additional therapeutic agent can be administered in thesame combination, e.g., parenterally, or the additional therapeuticagent can be administered as part of a separate composition or byanother method described herein.

Treatment with a dsRNA targeting SAA can also be performed incombination with radiation therapy, such as for treatment of a cancer,such as colon cancer or a carcinoma. A dsRNA featured herein may beadministered before or after a surgical procedure to treat a cancer(e.g., to remove a tumor, or a malignant cell or cell mass).

The invention features a method of administering a dsRNA targeting SAAto a patient having a disease or disorder mediated by SAA expression,such as AA amyloidosis. Administration of the dsRNA can stabilize andimprove renal function, for example, in a patient with AA amyloidosis.Patients can be administered a therapeutic amount of dsRNA, such as 0.5mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The dsRNAcan be administered by intravenous infusion over a period of time, suchas over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minuteperiod. The administration is repeated, for example, on a regular basis,such as biweekly (i.e., every two weeks) for one month, two months,three months, four months or longer. After an initial treatment regimen,the treatments can be administered on a less frequent basis. Forexample, after administration biweekly for three months, administrationcan be repeated once per month, for six months or a year or longer.Administration of the dsRNA can reduce serum SAA levels in the patientby at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

Before administration of a full dose of the dsRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction or a changein liver function. For example, in patients monitored for changes inliver function, a low incidence of LFT (Liver Function Test) change(e.g., a 10-20% incidence of LFT) is acceptable (e.g., a reversible,3-fold increase in ALT (alanine aminotransferase) and/or AST (aspartateaminotransferase) levels).

Methods for Inhibiting Expression of an SAA Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of an SAA gene in a mammal. The method includesadministering a composition featured in the invention to the mammal suchthat expression of the target SAA gene (e.g., one or both of SAA1 andSAA2) is silenced.

When the organism to be treated is a mammal such as a human, thecomposition may be administered by any means known in the art including,but not limited to oral or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal), intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by intravenousinfusion or injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the dsRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1 dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs are produced by solid phase synthesis on a scale of1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, AppleraDeutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG,500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNAand RNA containing 2′-O-methyl nucleotides are generated by solid phasesynthesis employing the corresponding phosphoramidites and 2′-O-methylphosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg,Germany). These building blocks are incorporated at selected siteswithin the sequence of the oligoribonucleotide chain using standardnucleoside phosphoramidite chemistry such as described in Currentprotocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.),John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkagesare introduced by replacement of the iodine oxidizer solution with asolution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents are obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC are carried out according to established procedures.Yields and concentrations are determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA is generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution is stored at −20° C. until use.

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as -Chol-3′), an appropriately modified solid support is used for RNAsynthesis. The modified solid support is prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) is added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) isadded and the mixture is stirred at room temperature until completion ofthe reaction is ascertained by TLC. After 19 h the solution ispartitioned with dichloromethane (3×100 mL). The organic layer is driedwith anhydrous sodium sulfate, filtered and evaporated. The residue isdistilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) is dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) is added to the solution at 0° C. It isthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution is brought to room temperature and stirred further for 6 h.Completion of the reaction is ascertained by TLC. The reaction mixtureis concentrated under vacuum and ethyl acetate is added to precipitatediisopropyl urea. The suspension is filtered. The filtrate is washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer is dried over sodium sulfate and concentrated togive the crude product which is purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) is dissolved in 20% piperidinein dimethylformamide at 0° C. The solution is continued stirring for 1h. The reaction mixture is concentrated under vacuum, water is added tothe residue, and the product is extracted with ethyl acetate. The crudeproduct is purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) is taken up in dichloromethane. Thesuspension is cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) is added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) isadded. The reaction mixture is stirred overnight. The reaction mixtureis diluted with dichloromethane and washed with 10% hydrochloric acid.The product is purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture is cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD is added slowly with stirring within 20 mins. The temperatureis kept below 5° C. during the addition. The stirring is continued for30 mins at 0° C. and 1 mL of glacial acetic acid is added, immediatelyfollowed by 4 g of NaH₂PO₄.H₂O in 40 mL of water The resultant mixtureis extracted twice with 100 mL of dichloromethane each and the combinedorganic extracts are washed twice with 10 mL of phosphate buffer each,dried, and evaporated to dryness. The residue is dissolved in 60 mL oftoluene, cooled to 0° C. and extracted with three 50 mL portions of coldpH 9.5 carbonate buffer. The aqueous extracts are adjusted to pH 3 withphosphoric acid, and extracted with five 40 mL portions of chloroformwhich are combined, dried and evaporated to dryness. The residue ispurified by column chromatography using 25% ethylacetate/hexane toafford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) is added drop wise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring is continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) is added, the mixture is extracted with ethylacetate (3×40mL). The combined ethylacetate layer is dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which ispurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicac id17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) is dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) are added withstirring. The reaction is carried out at room temperature overnight. Thereaction is quenched by the addition of methanol. The reaction mixtureis concentrated under vacuum and to the residue dichloromethane (50 mL)is added. The organic layer is washed with 1M aqueous sodiumbicarbonate. The organic layer is dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine is removed byevaporating with toluene. The crude product is purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) is mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture is dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) is added and thesolution is stirred at room temperature under argon atmosphere for 16 h.It is then diluted with dichloromethane (40 mL) and washed with ice coldaqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organicphase is dried over anhydrous sodium sulfate and concentrated todryness. The residue is used as such for the next step.

Cholesterol Derivatized CPG AI

Succinate AH (0.254 g, 0.242 mmol) is dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) are added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) is added. The reaction mixture turned brightorange in color. The solution is agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) isadded. The suspension is agitated for 2 h. The CPG is filtered through asintered funnel and washed with acetonitrile, dichloromethane and ethersuccessively. Unreacted amino groups are masked using aceticanhydride/pyridine. The achieved loading of the CPG is measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) is performed as described in WO2004/065601, except that, for the cholesteryl derivative, the oxidationstep is performed using the Beaucage reagent in order to introduce aphosphorothioate linkage at the 5′-end of the nucleic acid oligomer.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 1.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) Aadenosine-5′-phosphate C cytidine-5′-phosphate G guanosine-5′-phosphateT, dT 2′-deoxy-thymidine-5′-phosphate U uridine-5′-phosphate N anynucleotide (G, A, C, or T) a 2′-O-methyladenosine-5′-phosphate c2′-O-methylcytidine-5′-phosphate g 2′-O-methylguanosine-5′-phosphate u2′-O-methyluridine-5′-phosphate sT, sdT2′-deoxy-thymidine-5′phosphate-phosphorothioate

Example 2 siRNA Design

siRNA design was carried out to identify siRNAs targeting SAA1 and SAA2.The design used the SAA1 transcript NM_(—)000331.3 (human),NM_(—)009117.3 (mouse), and the single, near full length, SAA-like tracefrom cynomolgus monkey, the cDNA sequence Mfa#S27795076 (Macacafascicularis). The SAA2 transcripts used in designing the siRNAsincluded NM_(—)030754.2 (human) and NM_(—)011314.1 (mouse).

siRNA duplexes were designed with 100% identity to both SAA1 and SAA2genes. Several sets cross-reactive with human and mouse, human andcynomolgus monkey and human-cynomolgus monkey were designed.

All possible 19mers were created from each sequence. Human-mouse,human-cynomolgus monkey, and human-cynomolgus monkey-mouse subsets werecreated by searching for identical 19mers from each species using thePython script polyFastaToNmer.py. There were 254 human sense 19mersiRNAs. Of these 254, there were 21 with 100% identity to the human andmouse transcripts; 78 had 100% identity in human and cynomolgus monkey,and two had 100% identity in the three species (human, cynomolgusmonkey, and mouse).

The predicted specificity of each siRNA design was used as a criterionfor final selection. The SAA siRNAs were used in a comprehensive searchagainst the mouse, human and cynomolgus transcriptomes using the FASTAalgorithm. A Python script was then used to parse the alignments andgenerate a score based on the position and number of mismatches betweenthe siRNA and any potential ‘off-target’ transcript. The score isweighted to emphasize differences in the ‘seed’ region of siRNAs, inpositions 2-9 from the 5′ end of the molecule. Both siRNAs strands wereassigned to a category of specificity according to the calculatedscores: a score above 3 qualifies as highly specific, equal to 3 asspecific and between 2.2 and 2.8 as moderate specific.

Approximately 500-700 19-mer SAA siRNAs were designed and analyzed forSAA isoform/species cross-reactivity and off-target prediction. 78dsRNAs were selected for further analysis. These 78 siRNAs werepredicted to target both SAA1 and SAA2 (hereafter called “SAA” or“SAA½”). All 78 sense and antisense human-cynomolgus monkey-specificsiRNAs were synthesized with internal 2′Ome modifications and formedinto duplexes.

Example 3 In Vitro Efficacy Screening of Serum Amyloid A (SAA)

The 78 SAA siRNAs with 2′OM endo light modifications were screened forefficacy in an in vitro model. SAA-siRNA were reverse transfected at aconcentration of 20 nM in Hep3B cells using LF-Max. 24 h later, SAA wasinduced by adding combined IL-1β and IL6 cytokines. 18 h post-induction,SAA siRNA activity was analyzed by measuring the mRNA level by bDNA 2.0and TaqMan assays. Protein levels were measured using ELISA assays. Theresults, shown in Table 3, are from two biologicals, and two technicalrepeats.

Material and Methods:

Cell Culture:

Hep3B Cells (HB-8064™) were maintained at 37° C., 5% CO₂ in Eagle'sMinimum Essential Medium (EMEM-GIBCO) with 10% FBS (Omega ScientificCat#FB02) 1% Antibiotics/Antibiotics Cat#15240-062).

For stock culture, cells should be 90-100% confluent before splitting.Cells are washed and trypsinized with 3 ml 0.25% Trypsin-EDTA andincubated at 37° C., 5% CO₂. 7 ml of DMEM 10% FBS 1%Antibiotics/Antimicotics are added and the cells resuspended thoroughly.Appropriate aliquots of cells are added to a new flask containing 30 mlof fresh DMEM 10% FBS 1% Antibiotics/Antimicotics to obtain 90-100%confluence on the desired day. Cells are resuspended and incubated at37° C., 5% CO₂.

Reverse Transfection Using Lipofectamine RNAiMAX.

Lipofectamine™ RNAiMAX No. 13778-150 (1.5 ml size) was stored at +4° C.,as suggested by the manufacturer.

Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) was used to diluteRNAi duplexes and Lipofectamine™ RNAiMAX before complexing.

BLOCK-IT™ Alexa Fluor® Red Fluorescent Oligo (Cat. No. 14750-100) wasused for assessing transfection efficiency.

The Reverse Transfection procedure was used to transfect siRNA intoHep3B cells in a 96-well format. In reverse transfections, the complexeswere prepared inside the wells, after which cells suspension was added.For each well to be transfected, RNAi duplex-Lipofectamine RNAiMAXcomplexes were prepared as follows:

2 ul siRNA duplex (from 20 uM stock) were diluted in 198 μl Opti-MEM® ineach well of the dilution plate to have 20 nM final conc. For one dosescreening. For IC50 determinations, further dilutions were conducted bymix gently the previous dill and dilute 5 fold serially (40 ul from the1^(st) dill+160 ul OPT-MEM) to reach a range of 20 nM-50 fM final RNAconc. 10 ul/well siRNA dilution was transferred to the culture plate.

Lipofectamine™ RNAi MAX was mixed gently, then 20 ul Lipofectamine™ RNAiMAX was added to 10 ml Opt-MEM (0.2 μl Lipofectamine™/Well). 10 ul ofthe mixture was added to each well in the culture plate. The solutionwas mixed gently and incubated for 10-20 minutes at RT (20 ullipoplex/well).

The cells were split, counted and diluted in complete growth mediumwithout antibiotics so that 80 μl contained the appropriate number ofcells (2×10⁴/well) to give 30-50% confluence 24 hours after plating.

80 μl of the diluted cells were added to each well with RNAiduplex-Lipofectamine™ RNAiMAX complexes. This gave a final volume of 100μl and a final RNA concentration of 20 nM (for the single dose assay)and 20 nM-50 fM (IC50 assay). The cells were mixed gently by rocking theplate back and forth. The cells were incubated overnight at 37° C. in aCO2 incubator.

Human SAA Induction in Hep-3B Using Cytokines (IL-1β+IL-6).

500 ul deionized water was added to a vial of recombinant human IL-1β((rIL-1β, Thermo Scientific), ED50 or 1 unit of activity=3 pg/ml) toprepare a working stock solution. This dilution resulted in 20000000pg/500 ul=13333333 units total in 500 ul, and 1 ul stock solution=26666units

500 ul deionized water was added to a vial of Recombinant human IL-6(RIL-61, Thermo Scientific, ED50 or 1 unit of activity=52.5 pg/ml) toprepare a working stock solution. This dilution resulted in 20000000pg/500 ul=760456.3 units total in 500 ul, and a 1 ul stock solution=1521units.

Growth media with antibiotics was prepared in a volume sufficient foruse in the assay plates, where each well received 100 ul of the media.

For each 4 ml media, 1.2 ul (800 units/well) IL-1β working stocksolution and 0.6 ul (23 units/well) IL-6 working stock solution wasadded, and the solution was mixed well.

The media was removed from each plate and 100 ul of induction media wasadded. Some plates were incubated for 16-18 h at 37 C, 5% CO₂ to for usein the bDNA and TaqMan assays, and other plates were incubated for 44-46h for use in the ELISA assays.

FIGS. 1A and 1B show that SAA can be detected on both mRNA and proteinlevels. The combination of IL-1β and IL-6 caused a 12-14 fold increasein SAA mRNA levels after 16-18 hours, as measured by TaqMan assay, andcaused an 8-fold increase in SAA protein levels after 44 hours, asmeasured by ELISA.

Human SAA-bDNA Assay Using QuantiGene 2.0 Reagent System (Panomics).

The QuantiGene 2.0 bDNA assay was used to measure SAA RNA levels. AllbDNA probe sets were from Panomics. Human specific SAA probe sets weredesigned to detect both SAA-1 and SAA2 transcripts. Human GAPDH was usedas a control housekeeping gene, and the QuantiGene Kit (Panomics) wasused to run the assay.

1^(st) bDNA Day.

A fresh lysis mixture (Panomics) dilution was prepared by re-dissolvingany precipitates by incubating the mixture at 37° C. followed by gentleswirling. A 1:2 dilution was then prepared (1 volume of Lysis mixtureplus 2 volumes nuclease-free water). 10 ul/mL proteinase K (Panomics)was then added to the dilution. Cells were lysed 18 h post induction torelease the target RNA by first removing the supernatant from the cells,then adding 200 ul diluted lysis mixture plus Proteinase K. The plateswere sealed with aluminum tape and incubated 30-40 min at 55 C.

To capture target RNA from the cultured cells, plates were removed from4° C. storage and placed on the bench top to warm completely to roomtemperature. The sealed foil pouch was removed from the capture plateand 80 ul/well cell lysate was prepared for SAA analysis wells, while 80ul/well of 1:20 diluted lysate (in diluted lysis mixture 1:2) wasprepared for GAPDH analysis.

A working probe mix was prepared for SAA analysis and for GAPDH analysisin separate tubes (1.626 ul nuclease free H2O+887 ul lysis Mixture+134ul Blocking+40 ul 2.0 probe set=2.688 ul/plate). 20 ul/well of theworking probe mix was added, and the plates were sealed very tightly.The plates were incubated at 55 C overnight for hybridization.

2^(nd) bDNA Day.

1× wash buffer was prepared by adding 1.5 mL Wash Buffer Component 1 and2.5 mL Wash Buffer Component 2, to 496 mL nuclease-free water.

2.0 Pre-Amplifier working reagent was prepared by thawing 2.0Pre-Amplifier, and centrifuging briefly to collect the contents at thebottom of the tube. 11 μL, of the 2.0 Pre-Amplifier was added to 11 mLof Amplifier/Label Probe Diluent and the solution was inverted to mix.The solution was kept at room temperature until use.

2.0 Amplifier working reagent was prepared by thawing 2.0 Amplifier andthen centrifuging briefly to collect the contents at the bottom of thetube. 11 μL, of 2.0 Amplifier was added to 11 mL of Amplifier/LabelProbe Diluent and the solution was inverted to mix. This reagent wasalso kept at room temperature until ready for use.

2.0 Label Probe Working reagent was prepared by thawing 2.0 Label Probe,then centrifuge briefly to collect the contents at the bottom of thetube. 11 μL, of 2.0 0 Label Probe was added to 11 mL of Amplifier/LabelProbe Diluent and the solution was inverted to mix. This solution wasalso kept at room temperature until ready for use.

The 2.0 substrate was removed from storage at 4 C and allowed to warm toroom temperature before use.

200 ul/well of 1× Wash Buffer is added to the Capture plate, and theCapture Plate is inverted over an appropriate receptacle and thecontents are forcibly expelled. The inverted plate was firmly tapped ona clean paper towel to dry, and the wash was repeated two more timesusing 300 μL/well of 1× Wash. The plate was centrifuge at 240×g for 1min at room temperature.

For hybridization of the 2.0 Pre-Amplifier, 100 ul/well Pre-AmplifierWorking Reagent was added to the plate, and the plate was sealedtightly. The plate was then incubated at 55 C for 1 h. The plate waswashed three times after pre-amplification.

For hybridization of 2.0 Amplifier, Add 100 ul/well Amplifier WorkingReagent 100 ul/well was added to the plate. The plate was sealed verytightly and then incubated at 55 C for 1 h. The plate was washed threetimes after amplification.

For hybridization of the label Probe, 100 ul/well label Probe WorkingReagent was added to the plate, and the plate was sealed very tightly.The plate was incubated at 50 C for 1 h. The plate was washed threetimes after labeling.

For signal detection, 100 ul of 2.0 Substrate was added to each well andthe plate was read in the luminometer after 5 to 15 min.

Human SAA-TaqMan Gene Expression Assay (Applied Biosystems).

The Taqman assay used to measure SAA-RNA. All Taqman probes used for theTaqman assays were purchased from Applied Biosystems. The ABI 7900 HTand 7000 cyclers were used for processing and reading of assay plates.No RT PCR control should be run to check for any unspecificamplification or DNA contamination of the RNA used for the ReverseTranscription step.

The master mix was prepared by combining 10 μl PCR Gene ExpressionMaster mix (Applied Biosystems, ABI), 6 μl of Nuclease-free Water, 1 ulSAA probe designed to detect both SAA1 and SAA2 (Hs00761940_s1, AppliedBiosystems) and 1 ul from both 18 s endogenous control probe and 2 ul RTcDNA/add later for a total 20 ul reaction.

18 μl of the master mix was aliquoted into each well and then 2 μl ofcDNA RT product was added and mixed by pipetting up and down. The platewas sealed with AB Optic tape and processed with a Real Time PCRinstrument. Readings were taken on an ABI 7900 HT real time PCRinstrument after which data was analyzed and evaluated.

Human SAA-ELISA KIT Assay (Abazyme, LLC Cat #EL10015).

A Human SAA-ELISA assay was used to determine human serum amyloid A(SAA) protein in cell culture supernatant. Kit reagents were allowed toreach room temperature before using. An SAA Standard was reconstitutedwith 2.0 mL of Calibrator Diluent II (80 ng/ml)., and the solutionallowed to sit for at least 15 minutes with gentle agitation prior tomaking dilutions.

The stock solution was used to produce a serial 2-fold dilution serieswithin the range of the assay (2.5 ng/mL to 80 ng/mL). The undiluted SAAStandard served as the high standard (80 ng/mL) and the CalibratorDiluent served as the zero.

Supernatant samples from cells treated with siRNA and SAA induced oneday later by IL1b+IL-6 and after 46 post induction were used, andsupernates were diluted 1:3.

1× Wash Buffer (1:19 of distilled or deionized water) was prepared.Substrate A and Substrate B were mixed together in equal volumes 15minutes before use (need 14 ml total 7 ml each/plate).

To perform the assay, 100 μl of standard or sample were added to theappropriate well of a pre-coated microtiter plate with SAA specificmonoclonal antibody. The plate was covered and incubated for 1 hour atroom temperature. The plate was washed with 1× wash buffer (350 ul/well)five times. 100 μl of HRP-conjugate polyclonal antibody specific for SAAwas then added to each well. The plate was covered and incubated for onehour at room temperature, and the wash procedure was repeated fivetimes. 100 μL TMB (3,3′5,5′ tetramethyl-benzidine) substrate solutionwas added to each well. The plates were then covered and incubated for15 minutes at room temperature. An SAA and enzyme-substrate reactionexhibit a change in color.

The enzyme-substrate reaction was terminated by adding 100 μl stopsolution (sulphuric acid) to each well and mixing well. The OpticalDensity (O.D.) at 450±2 nm was measured within 30 minutes using aspectrophotometer (a microtiter plate) reader.

The SAA 2′-Ome duplex RNAs in Table 2 were synthesized; each strandincluded a phosphate link connecting adjacent 3′ dT molecules.

Symbols Used in Table 2

-   symbol definition-   A adenosine-3′-phosphate-   C cytidine-3′-phosphate-   G guanosine-3′-phosphate-   T 5-methyluridine-3′-phosphate-   U uridine-3′-phosphate-   c 2′-O-methylcytidine-3′-phosphate-   dT 2′-deoxythymidine-3′-phosphate-   u 2′-O-methyluridine-3′-phosphate    -   *Target is position of 5′ base on transcript of human SAA1        NM_(—)000331.3    -   Strand: S is sense; AS is antisense

TABLE 2 Sequences of SAA siRNAs Sequence without SEQ Sequence with  SEQModifications  ID Modifications ID AD ID # Strand Target* (5′-3′) NO:(5′-3′) NO: 18368 S 385 CCAUGCUCGGGGGAACUAU 157 ccAuGcucGGGGGAAcuAudTdT1 AS 403 AUAGUUCCCCCGAGCAUGG 158 AuAGUUCCCCCGAGcAUGGdTdT 2 18369 S 304GGCUUUUGAUGGGGCUCGG 159 GGcuuuuGAuGGGGcucGGdTdT 3 AS 322CCGAGCCCCAUCAAAAGCC 160 CCGAGCCCcAUcAAAAGCCdTdT 4 18370 S 285UCUUUUCGUUCCUUGGCGA 161 ucuuuucGuuccuuGGcGAdTdT 5 AS 303UCGCCAAGGAACGAAAAGA 162 UCGCcAAGGAACGAAAAGAdTdT 6 18371 S 352AGAAGCCAAUUACAUCGGC 163 AGAAGccAAuuAcAucGGcdTdT 7 AS 370GCCGAUGUAAUUGGCUUCU 164 GCCGAUGuAAUUGGCUUCUdTdT 8 18372 S 366UCGGCUCAGACAAAUACUU 165 ucGGcucAGAcAAAuAcuudTdT 9 AS 384AAGUAUUUGUCUGAGCCGA 166 AAGuAUUUGUCUGAGCCGAdTdT 10 18373 S 378AAUACUUCCAUGCUCGGGG 167 AAuAcuuccAuGcucGGGGdTdT 11 AS 396CCCCGAGCAUGGAAGUAUU 168 CCCCGAGcAUGGAAGuAUUdTdT 12 18374 S 551CCCAAUCACUUCCGACCUG 169 cccAAucAcuuccGAccuGdTdT 13 AS 569AGGUCGGAAGUGAUUGGGTT 170 cAGGUCGGAAGUGAUUGGGdTdT 14 18375 S 277CCGAAGCUUCUUUUCGUUC 171 ccGAAGcuucuuuucGuucdTdT 15 AS 295GAACGAAAAGAAGCUUCGG 172 GAACGAAAAGAAGCUUCGGdTdT 16 18376 S 359AAUUACAUCGGCUCAGACA 173 AAuuAcAucGGcucAGAcAdTdT 17 AS 377UGUCUGAGCCGAUGUAAUU 174 UGUCUGAGCCGAUGuAAUUdTdT 18 18377 S 361UUACAUCGGCUCAGACAAA 175 uuAcAucGGcucAGAcAAAdTdT 19 AS 379UUUGUCUGAGCCGAUGUAA 176 UUUGUCUGAGCCGAUGuAAdTdT 20 18378 S 383UUCCAUGCUCGGGGGAACU 177 uuccAuGcucGGGGGAAcudTdT 21 AS 401AGUUCCCCCGAGCAUGGAA 178 AGUUCCCCCGAGcAUGGAAdTdT 22 18379 S 386CAUGCUCGGGGGAACUAUG 179 cAuGcucGGGGGAAcuAuGdTdT 23 AS 404CAUAGUUCCCCCGAGCAUG 180 cAuAGUUCCCCCGAGcAUGdTdT 24 18380 S 305GCUUUUGAUGGGGCUCGGG 181 GcuuuuGAuGGGGcucGGGdTdT 25 AS 323CCCGAGCCCCAUCAAAAGC 182 CCCGAGCCCcAUcAAAAGCdTdT 26 18381 S 334AGCCUACUCUGACAUGAGA 183 AGccuAcucuGAcAuGAGAdTdT 27 AS 352UCUCAUGUCAGAGUAGGCU 184 UCUcAUGUcAGAGuAGGCUdTdT 28 18382 S 364CAUCGGCUCAGACAAAUAC 185 cAucGGcucAGAcAAAuAcdTdT 29 AS 382GUAUUUGUCUGAGCCGAUG 186 GuAUUUGUCUGAGCCGAUGdTdT 30 18383 S 547AGACCCCAAUCACUUCCGA 187 AGAccccAAucAcuuccGAdTdT 31 AS 565UCGGAAGUGAUUGGGGUCU 188 UCGGAAGUGAUUGGGGUCUdTdT 32 18384 S 579CUGAGAAAUACUGAGCUUC 189 cuGAGAAAuAcuGAGcuucdTdT 33 AS 597GAAGCUCAGUAUUUCUCAG 190 GAAGCUcAGuAUUUCUcAGdTdT 34 18385 S 275AGCCGAAGCUUCUUUUCGU 191 AGccGAAGcuucuuuucGudTdT 35 AS 293ACGAAAAGAAGCUUCGGCU 192 ACGAAAAGAAGCUUCGGCUdTdT 36 18386 S 286CUUUUCGUUCCUUGGCGAG 193 cuuuucGuuccuuGGcGAGdTdT 37 AS 304CUCGCCAAGGAACGAAAAG 194 CUCGCcAAGGAACGAAAAGdTdT 38 18387 S 287UUUUCGUUCCUUGGCGAGG 195 uuuucGuuccuuGGcGAGGdTdT 39 AS 305CCUCGCCAAGGAACGAAAA 196 CCUCGCcAAGGAACGAAAAdTdT 40 18388 S 357CCAAUUACAUCGGCUCAGA 197 ccAAuuAcAucGGcucAGAdTdT 41 AS 375UCUGAGCCGAUGUAAUUGG 198 UCUGAGCCGAUGuAAUUGGdTdT 42 18389 S 358CAAUUACAUCGGCUCAGAC 199 cAAuuAcAucGGcucAGAcdTdT 43 AS 376GUCUGAGCCGAUGUAAUUG 200 GUCUGAGCCGAUGuAAUUGdTdT 44 18390 S 299GGCGAGGCUUUUGAUGGGG 201 GGcGAGGcuuuuGAuGGGGdTdT 45 AS 317CCCCAUCAAAAGCCUCGCC 202 CCCcAUcAAAAGCCUCGCCdTdT 46 18391 S 316GGCUCGGGACAUGUGGAGA 203 GGcucGGGAcAuGuGGAGAdTdT 47 AS 334UCUCCACAUGUCCCGAGCC 204 UCUCcAcAUGUCCCGAGCCdTdT 48 18392 S 345ACAUGAGAGAAGCCAAUUA 205 AcAuGAGAGAAGccAAuuAdTdT 49 AS 363UAAUUGGCUUCUCUCAUGU 206 uAAUUGGCUUCUCUcAUGUdTdT 50 18393 S 346CAUGAGAGAAGCCAAUUAC 207 cAuGAGAGAAGccAAuuAcdTdT 51 AS 364GUAAUUGGCUUCUCUCAUG 208 GuAAUUGGCUUCUCUcAUGdTdT 52 18394 S 355AGCCAAUUACAUCGGCUCA 209 AgccAAuuAcAucGGcucAdTdT 53 AS 373UGAGCCGAUGUAAUUGGCU 210 UGAGCCGAUGuAAUUGGCUdTdT 54 18395 S 356GCCAAUUACAUCGGCUCAG 211 GccAAuuAcAucGGcucAGdTdT 55 AS 374CUGAGCCGAUGUAAUUGGC 212 CUGAGCCGAUGuAAUUGGCdTdT 56 18396 S 367CGGCUCAGACAAAUACUUC 213 cGGcucAGAcAAAuAcuucdTdT 57 AS 385GAAGUAUUUGUCUGAGCCG 214 GAAGuAUUUGUCUGAGCCGdTdT 58 18397 S 223CAUGAAGCUUCUCACGGGC 215 cAuGAAGcuucucAcGGGcdTdT 59 AS 241GCCCGUGAGAAGCUUCAUG 216 GCCCGUGAGAAGCUUcAUGdTdT 60 18398 S 485GGCCAUGGUGCGGAGGACU 217 GGccAuGGuGcGGAGGAcudTdT 61 AS 503AGUCCUCCGCACCAUGGCC 218 AGUCCUCCGcACcAUGGCCdTdT 62 18399 S 548GACCCCAAUCACUUCCGAC 219 GaccccAAucAcuuccGAcdTdT 63 AS 566GUCGGAAGUGAUUGGGGUC 220 GUCGGAAGUGAUUGGGGUCdTdT 64 18400 S 550CCCCAAUCACUUCCGACCU 221 ccccAAucAcuuccGAccudTdT 65 AS 568AGGUCGGAAGUGAUUGGGG 222 AGGUCGGAAGUGAUUGGGGdTdT 66 18401 S 573GCCUGCCUGAGAAAUACUG 223 GccuGccuGAGAAAuAcuGdTdT 67 AS 591CAGUAUUUCUCAGGCAGGC 224 cAGuAUUUCUcAGGcAGGCdTdT 68 18402 S 276GCCGAAGCUUCUUUUCGUU 225 GccGAAGcuucuuuucGuudTdT 69 AS 294AACGAAAAGAAGCUUCGGC 226 AACGAAAAGAAGCUUCGGCdTdT 70 18403 S 279GAAGCUUCUUUUCGUUCCU 227 GAAGcuucuuuucGuuccudTdT 71 AS 297AGGAACGAAAAGAAGCUUC 228 AGGAACGAAAAGAAGCUUCdTdT 72 18404 S 280AAGCUUCUUUUCGUUCCUU 229 AAGcuucuuuucGuuccuudTdT 73 AS 298AAGGAACGAAAAGAAGCUU 230 AAGGAACGAAAAGAAGCUUdTdT 74 18405 S 291CGUUCCUUGGCGAGGCUUU 231 cGuuccuuGGcGAGGcuuudTdT 75 AS 309AAAGCCUCGCCAAGGAACG 232 AAAGCCUCGCcAAGGAACGdTdT 76 18406 S 292GUUCCUUGGCGAGGCUUUU 233 GuuccuuGGcGAGGcuuuudTdT 77 AS 310AAAAGCCUCGCCAAGGAAC 234 AAAAGCCUCGCcAAGGAACdTdT 78 18407 S 296CUUGGCGAGGCUUUUGAUG 235 cuuGGcGAGGcuuuuGAuGdTdT 79 AS 314CAUCAAAAGCCUCGCCAAG 236 cAUcAAAAGCCUCGCcAAGdTdT 80 18408 S 298UGGCGAGGCUUUUGAUGGG 237 uGGcGAGGcuuuuGAuGGGdTdT 81 AS 316CCCAUCAAAAGCCUCGCCA 238 CCcAUcAAAAGCCUCGCcAdTdT 82 18409 S 340CUCUGACAUGAGAGAAGCC 239 cucuGAcAuGAGAGAAGccdTdT 83 AS 358GGCUUCUCUCAUGUCAGAG 240 GGCUUCUCUcAUGUcAGAGdTdT 84 18410 S 235CACGGGCCUGGUUUUCUGC 241 cAcGGGccuGGuuuucuGcdTdT 85 AS 253GCAGAAAACCAGGCCCGUG 242 GcAGAAAACcAGGCCCGUGdTdT 86 18411 S 306CUUUUGAUGGGGCUCGGGA 243 cuuuuGAuGGGGcucGGGAdTdT 87 AS 324UCCCGAGCCCCAUCAAAAG 244 UCCCGAGCCCcAUcAAAAGdTdT 88 18412 S 297UUGGCGAGGCUUUUGAUGG 245 uuGGcGAGGcuuuuGAuGGdTdT 89 AS 315CCAUCAAAAGCCUCGCCAA 246 CcAUcAAAAGCCUCGCcAAdTdT 90 18413 S 381ACUUCCAUGCUCGGGGGAA 247 AcuuccAuGcucGGGGGAAdTdT 91 AS 399UUCCCCCGAGCAUGGAAGU 248 UUCCCCCGAGcAUGGAAGUdTdT 92 18414 S 246UUUUCUGCUCCUUGGUCCU 249 uuuucuGcuccuuGGuccudTdT 93 AS 264AGGACCAAGGAGCAGAAAA 250 AGGACcAAGGAGcAGAAAAdTdT 94 18415 S 230CUUCUCACGGGCCUGGUUU 251 cuucucAcGGGccuGGuuudTdT 95 AS 248AAACCAGGCCCGUGAGAAG 252 AAACcAGGCCCGUGAGAAGdTdT 96 18416 S 360AUUACAUCGGCUCAGACAA 253 AuuAcAucGGcucAGAcAAdTdT 97 AS 378UUGUCUGAGCCGAUGUAAU 254 UUGUCUGAGCCGAUGuAAUdTdT 98 18417 S 379AUACUUCCAUGCUCGGGGG 255 AuAcuuccAuGcucGGGGGdTdT 99 AS 397CCCCCGAGCAUGGAAGUAU 256 CCCCCGAGcAUGGAAGuAUdTdT 100 18418 S 300GCGAGGCUUUUGAUGGGGC 257 GcGAGGcuuuuGAuGGGGcdTdT 101 AS 318GCCCCAUCAAAAGCCUCGC 258 GCCCcAUcAAAAGCCUCGCdTdT 102 18419 S 317GCUCGGGACAUGUGGAGAG 259 GcucGGGAcAuGuGGAGAGdTdT 103 AS 335CUCUCCACAUGUCCCGAGC 260 CUCUCcAcAUGUCCCGAGCdTdT 104 18420 S 324ACAUGUGGAGAGCCUACUC 261 AcAuGuGGAGAGccuAcucdTdT 105 AS 342GAGUAGGCUCUCCACAUGU 262 GAGuAGGCUCUCcAcAUGUdTdT 106 18421 S 384UCCAUGCUCGGGGGAACUA 263 uccAuGcucGGGGGAAcuAdTdT 107 AS 402UAGUUCCCCCGAGCAUGGA 264 uAGUUCCCCCGAGcAUGGAdTdT 108 18422 S 555AUCACUUCCGACCUGCUGG 265 AucAcuuccGAccuGcuGGdTdT 109 AS 573CCAGCAGGUCGGAAGUGAU 266 CcAGcAGGUCGGAAGUGAUdTdT 110 18423 S 322GGACAUGUGGAGAGCCUAC 267 GGAcAuGuGGAGAGccuAcdTdT 111 AS 340GUAGGCUCUCCACAUGUCC 268 GuAGGCUCUCcAcAUGUCCdTdT 112 18424 S 325CAUGUGGAGAGCCUACUCU 269 cAuGuGGAGAGccuAcucudTdT 113 AS 343AGAGUAGGCUCUCCACAUG 270 AGAGuAGGCUCUCcAcAUGdTdT 114 18425 S 330GGAGAGCCUACUCUGACAU 271 GGAGAGccuAcucuGAcAudTdT 115 AS 348AUGUCAGAGUAGGCUCUCC 272 AUGUcAGAGuAGGCUCUCCdTdT 116 18426 S 331GAGAGCCUACUCUGACAUG 273 GAGAGccuAcucuGAcAuGdTdT 117 AS 349CAUGUCAGAGUAGGCUCUC 274 cAUGUcAGAGuAGGCUCUCdTdT 118 18427 S 338UACUCUGACAUGAGAGAAG 275 uAcucuGAcAuGAGAGAAGdTdT 119 AS 356CUUCUCUCAUGUCAGAGUA 276 CUUCUCUcAUGUcAGAGuAdTdT 120 18428 S 353GAAGCCAAUUACAUCGGCU 277 GAAGccAAuuAcAucGGcudTdT 121 AS 371AGCCGAUGUAAUUGGCUUC 278 AGCCGAUGuAAUUGGCUUCdTdT 122 18429 S 369GCUCAGACAAAUACUUCCA 279 GcucAGAcAAAuAcuuccAdTdT 123 AS 387UGGAAGUAUUUGUCUGAGC 280 UGGAAGuAUUUGUCUGAGCdTdT 124 18430 S 380UACUUCCAUGCUCGGGGGA 281 uAcuuccAuGcucGGGGGAdTdT 125 AS 398UCCCCCGAGCAUGGAAGUA 282 UCCCCCGAGcAUGGAAGuAdTdT 126 18431 S 220CACCAUGAAGCUUCUCACG 283 cAccAuGAAGcuucucAcGdTdT 127 AS 238CGUGAGAAGCUUCAUGGUG 284 CGUGAGAAGCUUcAUGGUGdTdT 128 18432 S 410GCCAAAAGGGGACCUGGGG 285 GccAAAAGGGGAccuGGGGdTdT 129 AS 428CCCCAGGUCCCCUUUUGGC 286 CCCcAGGUCCCCUUUUGGCdTdT 130 18433 S 224AUGAAGCUUCUCACGGGCC 287 AuGAAGcuucucAcGGGccdTdT 131 AS 242GGCCCGUGAGAAGCUUCAU 288 GGCCCGUGAGAAGCUUcAUdTdT 132 18434 S 486GCCAUGGUGCGGAGGACUC 289 GccAuGGuGcGGAGGAcucdTdT 133 AS 504GAGUCCUCCGCACCAUGGC 290 GAGUCCUCCGcACcAUGGCdTdT 134 18435 S 487CCAUGGUGCGGAGGACUCG 291 ccAuGGuGcGGAGGAcucGdTdT 135 AS 505CGAGUCCUCCGCACCAUGG 292 CGAGUCCUCCGcACcAUGGdTdT 136 18436 S 237CGGGCCUGGUUUUCUGCUC 293 cGGGccuGGuuuucuGcucdTdT 137 AS 255GAGCAGAAAACCAGGCCCG 294 GAGcAGAAAACcAGGCCCGdTdT 138 18437 S 268UGUCAGCAGCCGAAGCUUC 295 uGucAGcAGccGAAGcuucdTdT 139 AS 286GAAGCUUCGGCUGCUGACA 296 GAAGCUUCGGCUGCUGAcAdTdT 140 18438 S 273GCAGCCGAAGCUUCUUUUC 297 GcAGccGAAGcuucuuuucdTdT 141 AS 291GAAAAGAAGCUUCGGCUGC 298 GAAAAGAAGCUUCGGCUGCdTdT 142 18439 S 282GCUUCUUUUCGUUCCUUGG 299 GcuucuuuucGuuccuuGGdTdT 143 AS 300CCAAGGAACGAAAAGAAGC 300 CcAAGGAACGAAAAGAAGCdTdT 144 18440 S 293UUCCUUGGCGAGGCUUUUG 301 uuccuuGGcGAGGcuuuuGdTdT 145 AS 311CAAAAGCCUCGCCAAGGAA 302 cAAAAGCCUCGCcAAGGAAdTdT 146 18441 S 294UCCUUGGCGAGGCUUUUGA 303 uccuuGGcGAGGcuuuuGAdTdT 147 AS 312UCAAAAGCCUCGCCAAGGA 304 UcAAAAGCCUCGCcAAGGAdTdT 148 18442 S 583GAAAUACUGAGCUUCCUCU 305 GAAAuAcuGAGcuuccucudTdT 149 AS 601AGAGGAAGCUCAGUAUUUC 306 AGAGGAAGCUcAGuAUUUCdTdT 150 18443 S 549ACCCCAAUCACUUCCGACC 307 AccccAAucAcuuccGAccdTdT 151 AS 567GGUCGGAAGUGAUUGGGGU 308 GGUCGGAAGUGAUUGGGGUdTdT 152 18444 S 393GGGGGAACUAUGAUGCUGC 309 GGGGGAAcuAuGAuGcuGcdTdT 153 AS 411GCAGCAUCAUAGUUCCCCC 310 GcAGcAUcAuAGUUCCCCCdTdT 154 18445 S 373AGACAAAUACUUCCAUGCU 311 AGAcAAAuAcuuccAuGcudTdT 155 AS 391AGCAUGGAAGUAUUUGUCU 312 AGcAUGGAAGuAUUUGUCUdTdT 156

TABLE 3 Results from in vitro efficacy screen of SAA siRNAs % SAAactivity relative to unspecific SAA-siRNA control AD-ID#specific/cross-reactive ELISA bDNA TaqMan 18406 Human 100 93 97 18440Human 100 92 96 18372 Human 100 94 92 18402 Human 100 92 94 18408 Human100 91 93 18386 Human 100 89 95 18390 Human 100 89 95 18403 Human 100 8896 18437 Human 94 93 97 18376 Human 100 95 87 18438 Human 100 90 9318396 Human 100 85 97 18370 Human 100 94 85 18416 Human 97 92 90 18384Human 100 85 92 18409 Human 98 83 89 18388 Human 100 80 88 18387 Human96 79 90 18427 Human 92 82 90 18377 Human 100 68 90 18407 Human 91 79 8718385 Human 100 51 97 18432 Human 88 76 82 18375 Human 100 67 80 18429Human 86 76 82 18439 Human 80 69 80 18380 Human 98 41 79 18381 Human 8572 66 18404 Human 83 64 73 18371 Human 65 96 29 18442 Human 90 56 6018389 Human 75 86 39 18393 Human 73 88 32 18401 Human 80 67 58 18382Human 82 70 51 18395 Human 63 60 57 18405 Human 46 60 58 18369 Human 8543 51 18394 Human 0 83 47 18392 Human 100 32 0 18412 Human 36 52 4918435 Human 41 45 45 18398 Human 28 46 46 18428 Human 4 27 57 18411Human 17 33 50 18441 Human 52 32 9 18418 Human 3 15 30 18431 Human andcyno 100 89 97 18400 Human and cyno 100 86 92 18420 Human and cyno 10087 89 18397 Human and cyno 100 83 92 18374 Human and cyno 99 90 82 18415Human and cyno 99 83 89 18436 Human and cyno 97 80 85 18425 Human andcyno 92 75 87 18399 Human and cyno 92 75 85 18391 Human and cyno 68 9275 18414 Human and cyno 86 73 81 18443 Human and cyno 91 74 70 18419Human and cyno 88 67 80 18383 Human and cyno 63 71 68 18410 Human andcyno 63 49 54 18426 Human and cyno 57 34 55 18422 Human and cyno 16 2341 18424 Human and cyno 0 24 38 18433 Human and cyno 0 33 20 18423 Humanand cyno 0 0 0 18417 Human and mouse 95 77 83 18379 Human and mouse 9954 89 18373 Human and mouse 96 76 69 18368 Human and mouse 88 79 5418430 Human and mouse 54 49 72 18413 Human and mouse 28 51 53 18421Human and mouse 14 49 45 18378 Human and mouse 0 50 20 18444 Human, cynoand mouse 92 73 76 18445 Human, cyno and mouse 93 68 74

FIGS. 2 and 3 illustrate SAA mRNA and protein levels in Hep3B cellsfollowing administration of the candidate SAA siRNAs as described above.Thirteen of the tested siRNA showed >90% inhibition of mRNA levels, 30siRNA showed >80% inhibition, and 60 siRNA showed >50% inhibition. Morethan 30 of the 78 candidate siRNA reduced protein levels by >95%.

Thirty-two of the 78 siRNA were selected for dose response and PBMCcytokine characterization. Selection was based on activity in singledose response experiment and on cross reactivity across species in orderto assay duplexes with human only activity, human/cyno activity,human/mouse activity, and human/mouse/cyno activity. Dose responsecurves for selected siRNAs are shown in FIGS. 4A-4G.

Results of the 1st Round of SAA-siRNAs IC50s an In Vitro Model.

To identify the most potent SAA siRNAs, IC50 of 32 SAA siRNAs werescreened in an in vitro model at concentrations ranging from 20 nM to 50fM (5 fold serial dilutions). SAA-siRNA were reverse transfected inHep3B using LF-Max. 24 h later, SAA gene expression was induced byadding combined IL-1β and IL6 cytokines. 18 h post-induction, SAA siRNAactivity was analyzed by measuring SAA mRNA levels relative to anonspecific control (BLOCK-IT) using bDNA 2.0. The results of the firstround screen are shown in Table 4 below.

TABLE 4 Results of first round screen of SAA siRNAs in in vitro modelSAA-siRNA specific/ IC50 (nM) ID# cross-reactive bDNA 18402 Human 0.000318384 Human 0.0035 18403 Human 0.0052 18406 Human 0.0058 18386 Human0.0064 18376 Human 0.0301 18396 Human 0.0304 18372 Human 0.0547 18437Human 0.0687 18438 Human 0.0828 18408 Human 0.0925 18390 Human 0.149018370 Human 0.1697 18416 Human 0.2548 18440 Human 0.7700 18409 Human0.8412 18400 Human and cyno 0.0004 18431 Human and cyno 0.0151 18397Human and cyno 0.1558 18420 Human and cyno 0.1612 18399 Human and cyno0.2097 18415 Human and cyno 0.4249 18374 Human and cyno 0.5581 18425Human and cyno 1.3838 18414 Human and cyno 1.7319 18436 Human and cyno4.2058 18379 Human and mouse 0.0466 18373 Human and mouse 0.2614 18417Human and mouse 0.6534

Results of the 2nd Round of SAA-siRNA IC50s in an In Vitro Model.

To identify potent SAA siRNAs, IC50 of 32 SAA siRNAs were screened in anin vitro model at concentrations ranging from 20 nM to 50 fM (5 foldserial dilutions). SAA-siRNA was reverse transfected in Hep3B usingLF-Max. 24 h later, SAA was induced by adding combined IL-1β and IL6cytokines. 18 h post-induction, SAA siRNA activity was analyzed bymeasuring the mRNA level of SAA relative to a nonspecific control(AD-1955) using bDNA 2.0. The results of the second round screening areshown below in Table 5. The shaded siRNAs in Table 5 were selected forfurther analysis in serum stability assays, in in vivo efficacy studiesin mice, and for off-target effects. Selection was based on the bestIC50 in each class of cross-reactivities; human/cyno was weightedheavier as it was the likeliest to produce a lead molecule, as moleculesin this class had great IC50 and would allow preclinical testing in NHP.

TABLE 5 Results of second round screen of SAA siRNAs in in vitro model

Example 4 In Vivo Mouse Model for Testing SAA siRNAs

An in vivo mouse model for testing SAA siRNAs was established. Mice(n=5) were injected intraperitoneally (i.p.) with lipopolysaccharide(LPS) at a concentration of 50 ug on day 0. Mice were bled on day −3 andday 1 following LPS injection and relative mouse SAA OD levels weremeasured.

FIG. 5 shows that SAA levels were increased in all mice tested 24 hoursafter LPS injection compared to pre-LPS injection SAA levels. SimilarSAA upregulation was achieved with 10 ug of LPS injected i.p. (data notshown).

To test whether SAA siRNA can downregulate SAA levels in vivo, mice wereadministered siRNA i.v. 6 hours after LPS injection (10 ug i.p.). ThesiRNAs tested were LNP01 formulated 18445 (10 mg/kg), LNP01 formulated18379 (10 mg/kg), and SNALP formulated 18445 (2 mg/kg). Controlsincluded PBS and LNP01 formulated 1955 control siRNA (10 mg/kg). Micewere bled 24 hours after siRNA administration and SAA levels weremeasured using ELISA assay.

SNALP formulation was as follows:: DLinDMA/DPPC/Cholesterol/PEG-cDMA(57.1/7.1/34.4/1.4) with a lipid:siRNA of ˜7:1.

LNP01 formulation was as follows: ND98/Cholesterol/PEG-Ceramide C16 witha 42:48:10 molar ratio.

FIG. 6 shows that LNP01-18445 and SNALP-18445 significantlydown-regulated SAA levels compared to controls.

As described in Table 2, the sequences of each strand of 18445 are asfollows:

   Sequence without SEQ Sequence with  SEQ Modifications  IDModifications ID AD-ID # Strand Target* (5′-3′) NO: (5′-3′) NO: 18445 S373 AGACAAAUACUUCCAUGCU 311 AGAcAAAuAcuuccAuGcudTdT 155 AS 391AGCAUGGAAGUAUUUGUCU 312 AGcAUGGAAGuAUUUGUCUdTdT 156

Alternative dsRNA are included in the invention, e.g., comprising atleast 15 nucleotides of the following sense or antisense strands:

SEQ ID  Strand Sequence (5′-3′) NO: S AGACAAAUACUUCCAUGCUNN 313 ASAGCAUGGAAGUAUUUGUCUNN 314 S AGAcAAAuAcuuccAuGcu 315 ASAGcAUGGAAGuAUUUGUCU 316 S AGAcAAAuAcuuccAuGcudTsdT 317 ASAGcAUGGAAGuAUUUGUCUdTsdT 318

Example 5 Animal Models for Testing SAA siRNAs

Endogenous mouse models are not suitable for testing human SAAsilencing. Therefore, SAA siRNAs can be tested in mice expressing humanSAA1 or SAA2 from a plasmid and/or from adenovirus.

An adenovirus expressing hSAA1 was engineered with a CMV immediate earlypromoter and enhancer to drive expression of hSAA1 (Hosai et al., JLR1999). Mice were pre-bled and then administered 4-12×10⁹ pfu/mouse. Micewere then bled on days 4, 8, 11, 15, and 22 following the virusadministration on day 0. FIG. 7 shows that expression of hSAA1 can lastfor approximately 2 weeks after a single injection of virus.

Hydrodynamic injection was also be used to express human SAA genes inmice (Nguyen et al., J. Surg. Res., 148:1, July 2008, p. 60-66; andHerweijer et al., J. Gene Med., 3:3, 2001, p. 280-291). A construct wasdesigned for hepatocyte-specific hSAA1 expression in mice (FIG. 8). Mice(n=3) were injected via tail vein with 50 ug of the construct plasmid inapproximately 2 ml of saline solution in approximately 10 seconds. Theexpression of hSAA1 in mice following hydrodynamic injection is shown inFIG. 9.

siRNAs can also be tested in mice expressing human SAA1 or SAA2 from atransgene. Transgenic mice can express the human SAA gene constitutivelyand for a longer period of time. A construct that was designed for hSAA1transgene expression is shown in FIG. 10 (Postic and Magnuson, Genesis,2000 February; 26(2):149-150.).

siRNAs can be tested in non-human primate (NHP) models using endogenousSAA expression. Reagents to detect NHP SAA mRNA and protein levels arevalidated, and then levels of circulating SAA in resting and diseasestates are determined before administering the candidate siRNAs.

Example 6 Inhibition of SAA Expression in Humans

A human subject is treated with a dsRNA targeted to a SAA gene toinhibit expression of the SAA gene for an extended period of timefollowing a single dose to treat a condition.

A subject in need of treatment is selected or identified. The subjectcan have AA amyloidosis, rheumatoid arthritis, a neoplasm, psoriaticarthritis, chronic juvenile arthritis, ankylosing spondylitis, Behcet'ssyndrome, Reiter's syndrome, adult Still's disease, inflammatory boweldisease, hereditary periodic fevers, tuberculosis, osteomyelitis,bronchiectasis, leprosy, pyelonephritis, decubitus ulcers, Whipple'sdisease, acne conglobata, common variable immunodeficiencyhypo/agammaglobulinemia, cystic fibrosis, hepatoma, renal carcinoma,Castleman's disease, Hodgkin's disease, adult hairy cell leukemia,Waldenström's disease, a neoplasm, a chronic infections, a chronicinflammatory disease, chronic arthritis, chronic sepsis, a periodicfever syndrome, familial Mediterranean fever, or Crohn's disease.

The identification of the subject can occur in a clinical setting, orelsewhere, e.g., in the subject's home through the subject's own use ofa self-testing kit.

At time zero, a suitable first dose of an anti-SAA siRNA issubcutaneously administered to the subject. The dsRNA is formulated asdescribed herein. After a period of time following the first dose, e.g.,7 days, 14 days, and 21 days, the subject's condition is evaluated,e.g., by measuring temperature or one or more inflammation biomarkers.This measurement can be accompanied by a measurement of SAA expressionin said subject, and/or the products of the successful siRNA-targetingof SAA mRNA. Other relevant criteria can also be measured. The numberand strength of doses are adjusted according to the subject's needs.

After treatment, the subject's temperature and/or inflammationbiomarker(s) are lowered relative to the levels existing prior to thetreatment, or relative to the levels measured in a similarly afflictedbut untreated subject.

Other embodiments are in the claims.

1. A double-stranded ribonucleic acid (dsRNA), wherein said dsRNAcomprises a sense strand and an antisense strand comprising a region ofcomplementarity which is complementary to at least a part of a mRNAencoding Serum amyloid A (SAA), and wherein said region ofcomplementarity is less than 30 nucleotides in length.
 2. The dsRNA ofclaim 1, wherein the dsRNA comprises a sense strand comprising at least15 contiguous nucleotides of a sense strand sequence selected from Table2.
 3. The dsRNA of claim 1, wherein the dsRNA comprises an antisensestrand comprising at least 15 contiguous nucleotides of an antisensesequence selected from Table
 2. 4. The dsRNA of claim 1, wherein thesense strand comprises 15 or more contiguous nucleotides of thenucleotide sequence of SEQ ID NO:311, SEQ ID NO:155, SEQ ID NO:37, SEQID NO:127, SEQ ID NO:95, SEQ ID NO:105, SEQ ID NO:59, SEQ ID NO:23, SEQID NO:193, SEQ ID NO:283, SEQ ID NO:251, SEQ ID NO:261, SEQ ID NO:215,or SEQ ID NO:179.
 5. The dsRNA of claim 1, wherein the antisense strandcomprises 15 or more contiguous nucleotides of the nucleotide sequenceof SEQ ID NO:312, SEQ ID NO:156, SEQ ID NO:38, SEQ ID NO:128, SEQ IDNO:96, SEQ ID NO:106, SEQ ID NO:60, SEQ ID NO:24, SEQ ID NO:194, SEQ IDNO:284, SEQ ID NO:252, SEQ ID NO:262, SEQ ID NO:216, or SEQ ID NO:180.6. The dsRNA of claim 1, wherein the sense strand consists of thenucleotide sequence of SEQ ID NO:311, SEQ ID NO:155, SEQ ID NO:37, SEQID NO:127, SEQ ID NO:95, SEQ ID NO:105, SEQ ID NO:59, SEQ ID NO:23, SEQID NO:193, SEQ ID NO:283, SEQ ID NO:251, SEQ ID NO:261, SEQ ID NO:215,or SEQ ID NO:179 and the antisense strand consists of SEQ ID NO:312, SEQID NO:156, SEQ ID NO:38, SEQ ID NO:128, SEQ ID NO:96, SEQ ID NO:106, SEQID NO:60, SEQ ID NO:24, SEQ ID NO:194, SEQ ID NO:284, SEQ ID NO:252, SEQID NO:262, SEQ ID NO:216, or SEQ ID NO:180.
 7. (canceled)
 8. The dsRNAof claim 1, wherein the dsRNA targets SEQ ID NO:311, SEQ ID NO:193, SEQID NO:283, SEQ ID NO:251, SEQ ID NO:261, SEQ ID NO:215, or SEQ IDNO:179.
 9. (canceled)
 10. The dsRNA of claim 1, where the mRNA encodesSAA1. 11.-13. (canceled)
 14. The dsRNA of claim 1, wherein said dsRNAcomprises at least one modified nucleotide.
 15. The dsRNA of claim 14,wherein at least one of said modified nucleotides is chosen from thegroup of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.
 16. ThedsRNA of claim 14, wherein said modified nucleotide is chosen from thegroup of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modifiednucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide. 17.-19.(canceled)
 20. The dsRNA of claim 1, wherein the dsRNA is conjugated toa ligand.
 21. The dsRNA of claim 1, wherein the dsRNA is formulated in alipid formulation. 22.-25. (canceled)
 26. The dsRNA of claim 1, whereinthe dsRNA has an IC50 of less than 10 pM. 27.-33. (canceled)
 34. A cellcontaining the dsRNA of claim
 1. 35. A pharmaceutical composition forinhibiting expression of an SAA gene comprising the dsRNA of claim 1.36. A method of inhibiting SAA expression in a cell, the methodcomprising: (a) introducing into the cell the dsRNA of claim 1; and (b)maintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of an SAA gene, therebyinhibiting expression of the SAA gene in the cell.
 37. A method oftreating a disorder associated with SAA expression comprisingadministering to a human in need of such treatment a therapeuticallyeffective amount of the dsRNA of claim
 1. 38. The method of claim 37,wherein the human has AA amyloidosis, rheumatoid arthritis, or aneoplasm.
 39. (canceled)
 40. (canceled)
 41. The method of claim 37,wherein the human has psoriatic arthritis, chronic juvenile arthritis,ankylosing spondylitis, Behcet's syndrome, Reiter's syndrome, adultStill's disease, inflammatory bowel disease, hereditary periodic fevers,tuberculosis, osteomyelitis, bronchiectasis, leprosy, pyelonephritis,decubitus ulcers, Whipple's disease, acne conglobata, common variableimmunodeficiency hypo/agammaglobulinemia, cystic fibrosis, hepatoma,renal carcinoma, Castleman's disease, Hodgkin's disease, adult hairycell leukemia, Waldenström's disease, a neoplasm, a chronic infections,a chronic inflammatory disease, chronic arthritis, chronic sepsis, aperiodic fever syndrome, familial Mediterranean fever, or Crohn'sdisease.
 42. A vector comprising a nucleotide sequence that encodes atleast one strand of the dsRNA of claim
 1. 43. The vector of claim 42,wherein the region of complementarity is at least 15 nucleotides inlength.
 44. (canceled)
 45. A cell comprising the vectors of claim 42.